Nucleic acids encoding FOXD3 promoter and methods to isolate FOXD3 expressing cells

ABSTRACT

DNA enhancer sequences are provided for use in constructs to identify early stage embryonic cells. The enhancer sequences can be used in parallel with short-hairpin RNA in a vector construct for endogenously regulated gene knockdowns. The disclosed enhancer sequences can be used to isolate a selected population of early stage embryonic cells.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. patent application Ser. No.12/645,431, filed Dec. 22, 2009 which claims the benefit of and priorityto U.S. Provisional Application Ser. No. 61/203,334, filed Dec. 22,2008, the entire contents of all of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. NS036485awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION BY REFERENCE

The material in the text file entitled “68663SEQLISTING” amended Sep.26, 2012 and being 82,374 bytes in size, is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Because of its stem cell properties and numerous derivatives, thevertebrate neural crest (NC) represents an excellent system forexamining questions of cell specification and differentiation duringdevelopment. A gene regulatory network (GRN) defines the regulatorystate of neural crest cells (Meulemans D & Bronner-Fraser M (2004) DevCell 7(3):291-299), such that modules of transcription factors functionsequentially to first specify the neural plate border and then thenascent neural crest. The intricate regulatory interactions within theNC-GRN start with a group of transcription factors comprising anevolutionarily “inflexible” neural plate border regulatory unit, whoseessential upstream function is to establish identity of the progenitorterritory (Nikitina N, Sauka-Spengler T, & Bronner-Fraser M (2008) ProcNatl Acad Sci USA 105(51):20083-20088). Neural crest specifiers aregenes responsible for formation of the neural crest. Sox10 is one of theearliest neural crest specifying genes, driving delamination anddirectly regulating numerous downstream effectors and differentiationgene batteries. FoxD3 is one of the first markers of pre-migratoryneural crest in many vertebrate species including mouse, chick, Xenopusand zebrafish (Hromas et al., 1999; Kos et al., 2001; Labosky andKaestner, 1998; Lister et al., 2006; Pohl and Knochel, 2001; Sasai etal., 2001; Yamagata and Noda, 1998). Identification of region-specificregulatory elements as described herein, provides an important tool foridentifying and manipulating the spatially-specified neural crest cells.

SUMMARY OF THE INVENTION

Identified and isolated DNA enhancer sequences are provided for use inconstructs to identify early stage embryonic neural crest cells. Theenhancer sequences can be used in parallel with short-hairpin RNA in avector construct for endogenously regulated gene knockdowns. Thedisclosed enhancer sequences can be used to isolate a selectedpopulation of early stage embryonic cells.

In a first aspect of the invention, an isolated DNA sequence isprovided, the sequence being selected from the group consisting of SEQID NOS: 1-6.

In a second aspect of the invention, an isolated DNA sequence isprovided, the sequence being selected from the group consisting of SEQID NOS: 7-12.

In a third aspect of the invention, a method is provided for isolating aselected group of cells from a population of cells, the methodcomprising: transfecting the population of cells with a DNA vectorconstruct having at least one enhancer sequence, wherein activation ofthe at least one enhancer sequence occurs in the selected population andactuates expression of a reporter protein; identifying the selectedgroup of cells; and collecting the selected population of cells byisolating cells expressing the reporter protein from cells which do notexpress the reporter protein.

In a fourth aspect of the invention, a DNA vector is provided fordown-regulating gene expression, the DNA vector comprising: ashort-hairpin RNA sequence under transcriptional control of at least oneenhancer sequence.

In a fifth aspect of the invention, a method is provided fordown-regulating gene expression using a DNA vector comprising ashort-hairpin RNA sequence under the transcriptional control of at leastone enhancer sequence, the method comprising: controlling transcriptionof the short-hairpin RNA sequence with endogenous factors that actuatethe at least one enhancer sequence.

In a sixth aspect of the invention, a method is provided fordown-regulating gene expression using a DNA vector comprising ashort-hairpin RNA sequence under transcriptional control of at least oneenhancer sequence, the method comprising: controlling transcription ofthe short-hairpin RNA sequence with endogenous factors that actuate theat least one enhancer sequence.

In a seventh aspect of the invention, an isolated DNA sequence isprovided, the sequence having 60%, 70%, 80%, 90% or 95% homology to asequence selected from the group consisting of SEQ ID NOS: 1-12.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic diagram of comparative genomic analysissurrounding the SOX10 gene in chicken, zebrafish, Xenopus, opossum,mouse, rat and human genomes.

FIG. 2 shows a table of primers used to amplify the at least 70%homologous regions across the compared genomes.

FIGS. 3B-3F show EGFP reporter expression activated by SOX10E.

FIGS. 3G-3J show in situ hybridization of endogenous Sox10.

FIGS. 4A-4F show spatially distinct expression of EGFP activated bySox10E1 and Sox10E2 in HH9, HH15 and HH18 embryos.

FIG. 5A shows homology of Sox10E1 and Sox10E2 across chicken, opossum,mouse, rat and human genomes.

FIGS. 5B-5D show Sox10E1- and Sox10E2-activated EGFP expression in HH9,HH15 and HH18 embryos.

FIG. 5E shows as schematic of a chick embryo with cranial, vagal andtrunk regions aligned with a table showing Sox10E1 and Sox10E2 activatedEGFP expression.

FIGS. 6A-6B show the region of Sox10E2 from the genomic comparison ofFIG. 5A.

FIG. 7A shows putative binding sites within Sox10E2.

FIGS. 7B-7F show Sox10E2-activated EGFP expression and the effect ofmutations in putative binding sites in Sox10E2.

FIGS. 8A-8E show Sox10E2-activated EGFP expression and the effect ofmutations in putative binding sites.

FIGS. 9A-9I show Sox10E2-activated EGFP expression in the presence ofmorpholinos to Ets1 and cMyb.

FIGS. 9J-9N show in situ hybridization of cMyb, Sox9 and Ets1 in HH6,HH8, and HH10 embryos.

FIGS. 10A-10F show Sox10E2-activated EGFP expression in the presence ofSox9 morpholinos.

FIGS. 11A-11H show that Sox9, cMyb and Ets1 morpholinos decreaseendogenous Sox10 expression.

FIGS. 11I-11M show rescue of the Sox9, cMyb or Ets1 knockdown of Sox10expression with co-expression of Sox9, cMyb or Ets1 DNA.

FIGS. 12A-12B show FITC labeled Ets1, cMyb and Sox9 morpholinos.

FIGS. 12C-12D show TUNEL staining.

FIGS. 12E-12F show anti-phospho histone H3 (PH3) antibody staining.

FIGS. 12G-12H show overlays of 12A-12F.

FIGS. 12I-12J show statistical calculations of stained cells.

FIGS. 13A-13J show that overexpression of Sox9, Ets1 or cMyb ectopicallyinduces Sox10E2-activated EGFP expression.

FIG. 13K shows agarose gel from EMSA assay.

FIG. 13L shows a Western blot of a DNA pulldown assay.

FIG. 13M shows results of in vivo qChIP assay.

FIGS. 14A-14F show ectopic Sox10E2-activated EGFP expression withmutations in binding sites in Sox10E2 and misexpression of Sox9, cMyband Ets1.

FIGS. 15A-15D show Western blots of cross-linked endogenous Ets1, cMyband Sox9.

FIGS. 16A-16D show calculated direct binding of Ets1, cMyb and Sox9 toSox10E2.

FIG. 17 shows a schematic diagram of comparative genomic analysissurrounding the FoxD3 gene in chicken, zebrafish, Xenopus, mouse, andhuman genomes.

FIGS. 18A-18M show in situ hybridization of FoxD3 and NC1-, NC2- andSC1-activated EGFP expression in chick embryos.

FIG. 19 shows a schematic of the sequence dissection of the NC1 enhancerregion.

FIGS. 20A-20J show EGFP expression activated by NC1.1 and NC1.2fragments of NC1 and Mut.7, Mut.8, and Mut.12 mutations of NC1.

FIGS. 21A-21G show EGFP expression activated by binding site mutationsin NC1.

FIG. 22 shows a schematic of the sequence dissection of the NC2 enhancerregion.

FIG. 23 shows a table of summarizing the expression of NC2 fragments.

FIG. 24A shows short-hairpin miRNA vector under the control of NC1.

FIG. 24B shows short-hairpin miRNA vector with human beta globin intronssurrounding the miRNA cloning site and an empty cloning site for anenhancer sequence.

FIG. 25 shows a schematic for the design and concept of short hairpinRNA vectors. Vector 2 differs from Vector 1 in that Vector 2 hassurrounded the microRNA with human beta globin intron sequence.

FIGS. 26A-26B show Sox100 knockdown by Sox100 shmiRNA under the controlof NC1 enhancer sequence.

FIG. 26C shows corresponding QPCR and FIG. 26D shows endogenous levelsof Sox10 mRNA by in situ hybridication.

FIGS. 27A-27B show EGFP tracer expression from the NC1-Sox10 shmiRNAvector of FIGS. 26A-26B.

FIG. 28A shows RFP in human ES cells infected with NC1-activated RFP;

FIG. 28B shows RFP in human ES cells infected with SOX10E-activated RFP.

DETAILED DESCRIPTION OF THE INVENTION

Dissection of the cis-regulatory regions of the essential neural crestspecifiers, Sox10 and FoxD3, has identified enhancer sequence regionswith distinct regulatory activities in the chick embryo. A Sox100enhancer region termed Sox100E (SEQ ID NO: 1) was identified andisolated. From dissection of the Sox100E region, Sox10E1 (SEQ ID NO: 2)and Sox10E2 (SEQ ID NO: 3) were identified and isolated. Three FoxD3enhancer regions termed NC (SEQ ID NO: 4), NC2 (SEQ ID NO: 5), and SC1(SEQ ID NO: 6) were identified and isolated.

The enhancer regions were identified using a comparative sequenceanalysis approach between multiple species. Sox10 and FoxD3 are genesimportant for formation, migration and differentiation, and aretherefore, highly conserved. Therefore, in principle, non-codingcis-regulatory regions are also conserved along with the genes theycontrol. As shown herein, the genomic sequences from chick, zebrafish,Xenopus, opossum, rat, mouse and human were compared. Conserved genomicregions isolated in this manner were amplified and subcloned intoreporter vectors, and the expression pattern of the reporter in vivo wastested using methods of in and ex ovo electroporation in chick embryos.The embryos were then cultured and allowed to developed for 12-24 hoursand subsequently examined for reporter activity.

As further described herein, the Sox10 and FoxD3 enhancer constructshave been subcloned into a variety of expression vectors thatsimultaneously express fluorescent proteins. These reporter constructscan be introduced into cells either by electroporation or by viralinfection using lenti virus. As shown herein, the enhancer constructsisolated from the chicken genome have been used to infect humanembryonic stem (ES) cells, wherein the reporter protein was activated incells directed to differentiate as human neural crest cells (FIGS. 28A,28B).

Because neural crest cells represent an important stem cell population,the enhancer constructs, as described herein, are important indicatorsof when a cell has acquired a neural crest fate and, thus, can beutilized in regenerative medicine laboratories for replacing neuralcrest derivatives. In one embodiment, the enhancer regions, as describedherein, are a tool for identifying and isolating neural crest cells. Inanother embodiment, the enhancer regions, when driving transcription ofa short-hairpin RNA expression vector, control targeted gene knockdownsto endogenous levels. In another embodiment, the enhancer regions areused to infect human embryonic stem (ES) cells, thereby providing ameans for directing human ES cells to differentiate.

Isolation of Sox10E. Sox10E1 and Sox10E2

Genomic sequences surrounding the Sox10 coding region from chicken,zebrafish, Xenopus, opossum, mouse, rat and human were compared insilico (FIG. 1), employing the ECR Browser program. Using Sox10 BACclone, genomic fragments of approximately 3-5 kilobases (kb), containingone or more conserved regions (≧70% homology) (FIG. 2) (SEQ ID NOS: 1,13-18), were cloned into an EGFP (enhanced green fluorescent protein)reporter vector upstream of thymidine kinase(tk) basal promoter(Uchikawa et al. (2003) Dev Cell 4(4):509-519) and functionally testedin vivo for its ability to recapitulate Sox10 expression during earlyneural crest formation. Using an ex ovo and in ovo electroporationtechniques (Sauka-Spengler and Barembaum (2008) Methods Cell Biol87:237-256), the entire epiblast of stage 4 (HH4) chick embryos,according to Hamburger and Hamilton (HH), or dorsal neural tube of stageHH8-12 embryos were transfected with reporter construct (green),together with a pCI-H2B-RFP (red) ubiquitous tracer to assesstransfection extent and efficiency. Embryos were collected after 8-48hours (HH8 to HH18), fixed and analyzed for EGFP expression.

The results reveal a 3.5 kb fragment, that is approximately 1 kbdownstream of the Sox10 coding region, that activates EGFP reporterexpression (FIGS. 3B-3F) in a manner that recapitulates endogenous Sox10transcription (FIGS. 3G-3J), as the neural crest delaminates andmigrates from the neural tube. EGFP transcripts were detected in cranialneural crest cells as early as HH8+ (FIG. 3B), in embryos with sixsomites, when Sox10 is first distinguishable by in situ hybridization(FIG. 3G). Both the EGFP reporter and endogenous Sox10 were maintainedon actively migrating cranial neural crest (FIGS. 3D, 3F, 3I) asexpression initiates progressively caudally (FIGS. 3I, 3J) (Cheng Y, etal. (2000) Brain Res Dev Brain Res 121(2):233-241.). However, whileendogenous Sox10 is down-regulated as crest cells enter the branchialarches (FIG. 3J), expression of the EGFP reporter was maintained inbranchial arches (similar to FIG. 4 b). Both Sox10 and EGFP were alsoexpressed in otic placode cells by stage HH10 (FIGS. 3C, 3H) and later,more caudally, in actively migrating, but not early delaminating vagaland trunk neural crest (FIGS. 3D, 3E, 3I, 3J).

Thus, this 3.5 kb Sox10 genomic fragment (denoted Sox10E) (SEQ ID NO: 1)contains regulatory modules that mediate initial Sox10 activation duringearly neural crest delamination at the cranial, but not more caudallevels. Of the six other fragments upstream of the coding region, fivefragments lacked functional activity at the time points of interest.Another 5 kb fragment, denoted Sox10L8 (FIG. 1), exhibited weak EGFPactivity in neural crest and otic cells by HH13 (6/6), but not inemigrating neural crest. (The ratio (6/6) refers to the number of timesthe result was observed over the number of times electroporation wasperformed. This ratio will be found throughout.)

Two highly conserved regions within Sox100E genomic fragment activatedistinct spatiotemporal reporter expression. The ECR browser program wasused to search for highly conserved sequences, potentially representingminimal essential core regulatory elements. By screening for 70%conservation across 100 bp windows within multiple aligned genomicregions between Sox10 and the first downstream neighboring gene, POLR2F,the program revealed two clusters of ˜160 base pairs (bp) and ˜267 bpwithin the 3.5 kb Sox10E fragment (SEQ ID NO: 1) (FIG. 5A). Assaying twosmaller fragments, each containing one identified conserved region,revealed that they activated EGFP expression in spatially distinctpopulations and in temporally distinct manners. A 600 bp fragmenttermed, Sox10E 1 (SEQ ID NO: 2), lacked activity in emigrating ormigrating cranial crest (FIGS. 4A-4D). It was first active in migratingvagal crest at HH15 (FIG. 5C) and in trunk crest, otic vesicle andcondensed trigeminal ganglia (FIGS. 5D, 5E), but did not drive EGFPexpression in delaminating vagal or trunk neural crest.

Systematic deletions within the Sox100E (SEQ ID NO: 1) region revealed asecond active region—a 264 bp minimal enhancer fragment, termed Sox10E2(SEQ ID NO: 2), comprised of an essential highly conserved 160 bp coreand supporting elements within 59 bp upstream thereof (FIGS. 6A-6B). Incontrast to the late activating Sox10E1, Sox10E2 displayed enhanceractivity as early as HH8+ in the first cranial crest emigrating from theneural tube, mimicking Sox10E activity (FIG. 3B) that intensifiedthrough HH9 (FIG. 5B). At HH12-15, Sox10E2 reporter expression wasmaintained in periocular crest, rostral hindbrain streams and oticvesicle (OV) (FIGS. 4B, 4E), but absent from caudal hindbrain or trunklevels (FIGS. 4B, 4C, 4E, 4F). Just as Sox10E (SEQ ID NO: 1) displaysregulatory activity within the branchial arches, Sox10E2 (SEQ ID NO: 3)drives EGFP expression in rostral hindbrain crest populating the firsttwo arches (FIGS. 4B, 4E), and Sox10E1 (SEQ ID NO: 2) is active invagally-derived crest (rhombomeres 6-8) (arrows, FIG. 4B) of posteriorbranchial arches 3-5 (arrowheads, FIG. 4D). In contrast, expression ofendogenous Sox10 is down-regulated upon entering the arches (FIG. 3J).This ectopic expression is indicative of a loss of a repressor elementfrom the Sox10E fragments. The results show that both cis-regulatoryfragments Sox10E1 (SEQ ID NO: 2) and Sox10E2 (SEQ ID NO: 3) can regulateSox10 expression in neural crest and otic regions, but in spatially andtemporally distinct patterns. Each Sox10E1 (SEQ ID NO: 2) and Sox10E2(SEQ ID NO: 3) regulates a portion of endogenous Sox10 expression, whichinitiates in a rostrocaudal temporal sequence (FIG. 5E).

Binding motifs for SoxE, Ets and Myb are necessary for Sox10E2regulatory activity. To identify putative transcription factor bindingsites within the 264 bp Sox10E2 regulatory fragment, the correspondingsequences from human, mouse, rat, opossum and Xenopus genomic regionswere aligned to chicken and screened for conserved motifs.Concomitantly, sequences were analyzed for known transcription factorconsensus sites using Transfac 7.0, rVista and Jaspar programs. Thisrevealed three highly conserved binding motifs (100% homology acrossamniotes), two for the SoxE family of proteins (Sox8, Sox9, Sox10) andone for Ets factors. Conservation of other putative binding motifsranged from 50-80% (FIG. 7A). Computationally identified binding motifswithin Sox10E2 were tested for function via mutation/deletion analyses.Mutated versions of Sox10E2-EGFP constructs were generated forindividual putative binding motifs, electroporated into chicken embryos,and analyzed after 10-12 hours (HH10-12).

Mutation of a putative Ets binding motif, within the enhancer core (M9;FIG. 7A), completely abolished Sox10E2 expression (FIG. 7C; 8/8).Similarly, reporter activity in cranial neural crest (FIG. 7B) waseliminated upon mutation of either SoxE binding site within theessential core region (M8,M11; FIG. 7A), indicating both were requiredfor its activity (FIG. 8 b; 13/13). Interestingly, there are twoputative binding motifs for Myb factors in Sox10E2, one within the coreand the other in the upstream adjacent supporting region (M2,M12; FIG.7A), each contributing to regulatory activity. When both were replacedwith random sequences, this double mutation completely abolishedreporter expression (FIG. 8C; 7/7). Individual mutation of othercomputationally identified motifs only reduced enhancer activity. Forexample, perturbations of SoxD (M13; 10/10), Elk/Ets(M4; 7/7) and singleMyb (M2,M12; 6/6) sites diminished EGFP signal intensity (FIGS. 7A,7D;FIG. 8D) suggesting they enhance regulatory function. In contrast,several mutations had no effect; e.g. simultaneous mutation of fourputative Pax binding sites (M1,M3,M5,M7; FIGS. 7A,7E; 7/7), deletion of45 bp within the core region (FIG. 7A, faded portion; 11/11), ormutation of either of two NFκB binding site (M6,M10; FIG. 8; 6/6). Takentogether, these results show that SoxE, Ets and Myb binding motifs areeach necessary for Sox10E2 regulatory function. In addition to neuralcrest expression, these mutations also affected expression of theSox10E2 reporter in the otic placode.

We tested whether SoxE, Ets and Myb binding sites, within the 264 bpSox10E2 fragment (SEQ ID NO: 3), are essential for regulatory activityof a larger construct from the Sox10 locus. To this end, we mutatedthese same sites (M2,M8,M9,M11,M12; FIG. 7A) within a much largergenomic fragment (˜3.5 kb) to test whether other genomic regionssurrounding these enhancers could compensate for the loss of activity.Whereas the full length, non-mutated construct gave robust GFP stainingthat recapitulated endogenous Sox10 expression, reporter activity indelaminating neural crest was completely eliminated in the sameconstruct bearing mutations in SoxE, Ets and Myb binding sites withinSox10E2 (FIG. 7F; 6/6). As expected, later reporter expression wasobserved in migrating vagal and trunk neural crest since the mutatedversion still contained an intact Sox10E1 enhancer. These resultsstrongly suggest that 264 bp Sox10E2 fragment (SEQ ID NO: 3) representsan essential regulatory module, and that binding sites for SoxE, Ets andMyb proteins are absolutely required for early Sox10 expression withinthe context of the Sox10 locus.

Knockdown of Ets1, cMyb or Sox9 diminishes Sox10E2 regulatory activity.To test if Ets1, cMyb and Sox9 transcription factors are required forexogenous Sox10E2 regulatory activity in delaminating neural crest, weco-electroporated either Ets1, cMyb, or Sox9 morpholino with the Sox10E2reporter construct. The right side of each embryo received morpholinoplus Sox10E2 reporter, whereas the left side received reporter plasmidalone. When the reporter construct was co-electroporated with controlmorpholino, reporter signal on the right side was unaffected andcomparable to the contralateral side (FIGS. 9A-9C; 10/10). Conversely,in the presence of cMyb (FIGS. 9D-9F; 11/15), Ets1(FIGS. 9G-9I; 13/15),or Sox9 morpholino (FIG. 10; 15/15) expression was greatly decreased orabolished. These results show that Ets1, cMyb and Sox9, areindependently required for the normal Sox10E2 regulatory activity,therefore making them good candidate factors responsible for the initialregulation of Sox10 through the identified Ets, Myb and SoxE functionalbinding motifs within Sox10E2.

Knockdown of Ets1, cMyb or Sox9 diminishes endogenous Sox10 expression.Although cMyb transcripts have been detected in early embryogenesis(Karafiat V, et al. (2005) Cell Mol Life Sci 62(21):2516-2525), theirdistribution was unknown and has not been described within the contextof the neural crest gene regulatory network. Our results, using in situhybridization, show that cMyb is expressed at stage HH6 in the neuralplate border (FIG. 9J), and that transcripts accumulate in the neuralfolds by HH8, with strongest expression at the dorsal margins containingneural crest precursors (FIGS. 9K, 9K′). At HH10, transcripts are seenin neural crest cells delaminating and emigrating from the cranialneural tube (FIGS. 9L, 9L′). Thus, cMyb, like Sox9 (FIG. 9M) and Ets1(FIG. 9N), is expressed in presumptive cranial neural crest prior toSox10. The presence of cMyb at the neural plate border and premigratoryneural crest illuminates a new role, at the onset of Sox10 expression,in neural crest cell specification. Its initial expression coincideswith that of early neural crest specifiers such as AP-2, c-Myc orSnail2. Furthermore, overexpression of cMyb up-regulates Msx1 andSnail2, and thus, participates in BMP4 input into theepithelial-mesenchymal transition of trunk neural crest (Karafiat V, etal. (2005) Cell Mol Life Sci 62(21):2516-2525).

In order to confirm that endogenous Ets1, Sox9 or cMyb proteins arerequired as upstream regulators of Sox10 in delaminating crest in vivo,the effects of cMyb, Ets1 or Sox9 morpholinos on endogenous Sox10expression at HH8+−9 was examined The results reveal a dose-dependenteffect on Sox10 expression on the electroporated versus contralateralside. A mild diminution was observed when individual morpholinos wereelectroporated at 1 mM (Sox9 3/3; cMyb 9/10; Ets1 7/10), but a markeddecrease at 3 mM (Sox9 n=5, cMyb and Ets1 n=6, p<0.02; FIGS. 1A-11C,11E-11G). The effect of cMyb knockdown was less strong than either Ets1or Sox9 inactivation (phenotypes ranging from 50-75% loss in Sox10transcript). In contrast, electroporation of a control morpholino had noeffect (FIGS. 11L, 11M; 10/10) and co-electroporation of morpholinoswith the corresponding mRNAs mutated within the morpholino target regionsuccessfully rescued the loss-of-function phenotype (Sox9, n=6, p<0.03;cMyb n=5, p≦0.04: Ets1, n=5, p<0.03: FIGS. 11I-11K). No statisticallysignificant differences were noted in phosphohistone H3 or TUNELstaining between electroporated and control sides of embryos receivingeither individual or all three morpholinos (˜3 mM). Thus, changes incell proliferation or cell death cannot account for loss of Sox10transcript (FIGS. 12A-12J). The cumulative results indicate that Sox9,cMyb and Ets1 are each required for expression of endogenous Sox10.Importantly, the combined electroporation of all three morpholinosvirtually eliminated transcript expression on the electroporated side(n=6, p≦0.01; FIGS. 12D,12H). As shown herein, Sox9, cMyb and Ets1together are necessary for initial activation of Sox10.

Sox9, Ets1 and cMyb ectopically activate and are required for Sox10E2reporter expression. All three SoxE genes, Sox8, Sox9 and Sox10 areexpressed by neural crest progenitors (Haldin C E & Labonne C (2009) IntJ Biochem Cell Biol.) Because these genes can act redundantly (Finzschet al. (2008) Development 135(4):637-646; Stolt et al. (2004)Development 131(10):2349-2358; Taylor and Labonne (2005) Dev Cell9(5):593-603), theoretically any could activate the Sox10E2 reporterconstruct within the endogenous context. In all vertebrates examined,however, Sox9 expression precedes Sox10 (Antonellis A, et al. (2006) HumMol Genet. 15(2):259-271; Dutton J R, et al. (2008) BMC Dev Biol 8:105;Werner et al. (2007) Nucleic Acids Res 35(19):6526-6538; Hong andSaint-Jeannet, (2005) Semin Cell Dev Biol 16(6):694-703.) e.g. chickSox9 is expressed in dorsal neural folds as early as HH8, before eitherSox10 or Sox8 (Cheung and Briscoe (2003) Development130(23):5681-5693.). This narrow (4-6 hr) time delay and the Sox9morpholino knock down results indicate that Sox9 directly regulatesSox10 onset and that this SoxE protein is responsible for initiatingSox10 expression. To test if Sox9 can regulate the identified Sox10E2regulatory element, Sox9 protein was ectopically expressed usingubiquitous H2B-RFP expression vector. Whereas no ectopic reporterexpression was seen when Sox10E2 reporter was co-electroporated withcontrol plasmid (FIGS. 13A, 13F; 9/9), co-electroporation with Sox9plasmid caused ectopic reporter activity in extra-embryonic region(FIGS. 13B,13G; 6/6). Similar results were obtained when cMyb wasectopically expressed (FIGS. 13D, 13I; 3/3). Because Sox9 is expressedonly transiently in migrating neural crest cells, it is likely thatSox10 and/or Sox8 later act to maintain Sox10 expression.

Co-electroporation of Ets1 plasmid with Sox10E2 reporter resulted inectopic reporter activation not only in extra-embryonic regions, butalso in the trunk neural tube, which normally does not express Ets1(Tahtakran and Selleck (2003) Gene Expr Patterns 3(4):455-458)(arrowheads; FIGS. 13C,13H; 12/12). In the embryo, Ets1 plays a role incranial neural crest delamination and appears to mitigate therequirement for S phase synchronization to promote crest emigration in acluster-like fashion. Moreover, ectopic expression of Ets1 in the trunkresults in excess, cluster-like emigration of Sox10-expressing cells(Theveneau et al. (2007) PLoS ONE 2(11): e1142). Since expression ofboth Ets1 and the Sox10E2-driven reporter is cranial-specific, Sox10E2acts like a switch distinguishing head and trunk crest populations.Since Sox9 and cMyb, but not Ets1, are normally expressed in the trunkneural tube (Tahtakran and Selleck, (2003) Gene Expr Patterns3(4):455-458; Karafiat V. et al. (2005) Cell Mol Life Sci62(21):2516-2525), Cheung and Briscoe, (2003) Development130(23):5681-5693), ectopic Ets1 in this location likely cooperates withthese other factors to induce reporter expression. To support this,combined overexpression of Sox9, Ets1 and cMyb has a broader effect andinduces strong ectopic Sox10E2 expression not only extra-embryonically,but also along the neural tube, and in the ectoderm (FIGS. 14A,14D;5/5).

Sox9, Ets1 or cMyb are each sufficient to trigger ectopic Sox10E2enhancer activity. However, mutation of individual binding motifs orknock down of individual factors in the endogenous context shows thatall three factors are necessary for normal Sox10E2 regulatory activity.Ectopic reporter activity driven by overexpression of individualtranscription factors occurs mainly in the extra-embryonic region. Thisresult indicates that these naïve, early stage cells already containregulatory factors characteristic of multipotent tissue and are, thus,competent to switch on a neural crest-like transcriptional program inresponse to the proper single inputs.

To test if regulatory activity is mediated via the corresponding bindingmotifs of Sox9, Ets1 and Myb within Sox10E2 enhancer, we assayed theirability to ectopically activate mutated reporter constructs. EitherSox9-H2B RFP (6/6) or cEts1-H2B RFP (6/6) were co-electroporated withSox10E2 construct with corresponding binding motif mutations. In allcases, electroporated embryos lacked ectopic reporter expression (FIGS.13E, 13J). However, ectopic reporter expression was also affected whenoverexpressing either Ets1, cMyb or Sox9 with other Sox10E2 versions,containing mutations within non-cognate biding sites. For example, whenSox9 and cMyb were overexpressed and combined with a Sox10E2 reportercarrying a mutation within the Ets motif (M9), ectopic reporterexpression in the extra-embryonic region was not observed (FIGS. 14B,14E; 6/6). If Sox9 and Ets1 were over-expressed together with a Sox10E2carrying a single mutated Myb site (M12), ectopic reporter expressionwas weak (FIGS. 14C. 14F; 3/3). This shows that for the Sox10E2 enhancerto have ectopic regulatory activity all binding sites need to befunctional and indicates a cluster-like conformation of the motifs andsynergistic action of the corresponding upstream regulators.

Sox9, Ets1 and cMyb directly bind to the Sox10E2 element. To determineif Sox9, Ets1 and cMyb can bind directly to the corresponding motifswithin the Sox10E2 element, EMSA assays were performed usingbiotinylated double stranded oligonucleotides containing thecorresponding Sox10E2 sub-fragments (underlined, FIG. 7A). A clearelectrophoretic shift was observed in samples incubated with nuclearextracts from chicken embryonic fibroblasts overexpressing Sox9, Ets1 orcMyb, but not from the cells transfected with control plasmid (whitearrowheads; FIG. 13K). This binding was out-competed by adding 200-foldexcess of the corresponding non-labeled (cold) fragment to the bindingreaction, showing specificity. The identity of the transcription factorsdirectly binding to Sox10E2 subfragments was confirmed using astreptavidin-biotin DNA pulldown approach followed by Western blot withspecific antibodies. Using biotinylated target and scrambled controlfragments as bait, it was shown that specific subfragments pull downcorresponding binding proteins (Sox9, Ets1 and cMyb) from the embryonicnuclear extracts. Conversely non-coated streptavidin-conjugated magneticbeads or beads coated with scrambled control fragments display nospecific protein binding (FIG. 13L).

Direct binding of these transcription factors to the Sox10E2 enhancerwas determined in vivo using quantitative ChIP(qChIP). Crosslinkedchromatin isolated from cranial regions of HH8-12 somite embryos wasimmunoprecipitated using Sox9, Ets1 and cMyb antibodies andChIP-enriched DNA was used in site-specific QPCR, with primers designedto amplify fragments within the Sox10E2 region. The results showsignificant (4-8×) enrichment over non-specific antibody indicating thatSox10 locus and, in particular, Sox10E2 regulatory element, was occupiedby endogenous Sox9, Ets1 and cMyb proteins in cranial region of HH8-HH10chicken embryos (FIG. 13M).

As shown in FIGS. 3B-16 d, Sox10E1 and Sox10E2 regulate different stagesof Sox10 expression. In one embodiment, Sox10E1 (SEQ ID NO: 2) is usedas a vector expression “driver” to identify cells or manipulate in vivoexpression (targeted gene knockdown using miRNA, morpholino, etc) atstage HH15. In another embodiment, Sox10E2 (SEQ ID NO: 3) is used as anvector expression “driver” to identify cells or manipulate in vivoexpression (at stages HH8-HH15. Cells or in vivo expression can beidentified or monitored using a number of methods known in the art—e.g.the “driven” or activated expression can be of any protein that can bemonitored. As shown herein, the EGFP and RFP proteins are expressed andthe fluorescence is imaged. The enhancer sequences can “drive” theexpression of any protein that can be monitored via fluorescence aloneif the expressed protein is a fluorescent protein, specific antibody tothe protein, or an antibody to a tag fused with the protein. Fluorescentexpression allows for the utilization of flow cytometry to specificallyisolate and sort those cells which are able to activate the particularenhancer region, from those which cannot.

Expression Patterns of FoxD3 Enhancers: NC1, NC2 and SC1

The genomic region of FoxD3 was examined for conservation across chick,mouse, human and also opossum, Xenopus and zebrafish. The region spanned160 kb between the genes immediately up and downstream of FoxD3, Atg4Cand Alg6 (FIG. 17). Conserved regions varying in size from 1 kb to 4 kbwere tested for enhancer activity between stages 8 and 14. Threeenhancers. NC1, NC2 and SC1, (SEQ ID NOS: 4-6) were found to drivespecific expression. All three enhancers are upstream of the FoxD3 gene.NC1 (SEQ ID NO: 4) is located 20 kb upstream; NC2 (SEQ ID NO: 5) islocated 44 kb upstream and SC1 (SEQ ID NO: 6) is located 24 kb upstream.

In situ hybridization of FoxD3 at stage 8 is shown in FIG. 18A.NC1-directed (SEQ ID NO: 4) expression of EGFP in the premigratorycranial neural crest beginning at stage 8 is shown in FIG. 18B. In situhybridization of FoxD3 in stage 11 embyros as shown in FIG. 18C.NC1-directed expression in stage 11 embryos continued during neuralcrest migration (FIG. 18D, 18G) and lasted until approximately stage 14,at which stage only very weak EGFP expression could be detected. Onlythe neural crest from the midbrain to rhombomere (R) 2 showed expressionfrom NC1 (FIG. 18D). Expression of NC-1-EGFP was not seen caudal to R3.

In contrast to NC-1, the enhancer NC2 (SEQ ID NO: 5) directed strongexpression of EGFP in the premigratory and migratory neural crest in R6and caudal to R6 (FIG. 18E). The expression pattern of EGFP in the vagaland trunk neural crest matched the mRNA expression of FoxD3, and bothextend to the premigratory crest at the level of the 4^(th) most caudalsomite. Expression of NC-2-activated EGFP in R4 crest was weaker, incontrast to the early expression in the vagal and trunk neural crest(FIG. 18E). There was also weak expression of EGFP in the migratingcranial neural crest (FIG. 18H), which was not detected prior to stage9+. The first expression of EGFP driven by NC2 was observed in the vagaland trunk levels of stage 9 embryos in pre-migratory neural crest.Additionally, very weak EGFP expression was also observed in thedeveloping optic retina from stage 11 and a very small number of cellsdisplayed very weak expression in the otic vesicle at stage 12.

To examine the temporal regulation of the NC2 enhancer (SEQ ID NO: 5),stage 8 to 14 embryos were electroporated using in ovo electroporation,and the embryos were fixed after 24-48 h. FoxD3 is expressed in mostpre-migratory and migratory vagal and trunk neural crest, but is notexpressed by pre-migratory and migratory melanoblasts, which undergoemigration approximately 24 h after the emigration of ganglionic neuralcrest. NC2-activated EGFP expression was observed in melanoblasts priorto and during migration (FIG. 18L), in addition to expression in thedorsal root ganglia. To confirm that this expression was due to activityof the enhancer and not stability of EGFP, in situ hybridization forEGFP was performed and mRNA for EGFP was detected in melanoblasts anddorsal root ganglia. Expression of NC-2-activated EGFP was also observedin neural crest cells migrating along the enteric nervous system.

The third FoxD3 enhancer, SC1 (SEQ ID NO: 6), directed expression ofEGFP in a subset of cells in the vagal and trunk neural tube from R5caudally beginning at stage 8+. These SC-1-activated EGFP-positive cellswere seen in the dorsal neural tube in the location of pre-migratoryneural crest, and also in at mid levels of the neural tube (FIG. 18F,18J). Some SC-1-activated EGFP-positive cells were seen emigrating fromthe neural tube, however expression was not seen in the dorsal rootganglia. FIG. 18M shows a cross-section of E4 embryo with EGFP driven bySC1 enhancer. Electroporation at stage 12 and fixation after 24 and 48 hrevealed no expression with SC1-activated EGFP in the dorsal rootganglia or other neural crest derivatives, but SC-1-activated EGFP wasseen in a population of interneurons in the neural tube at E4, whichcorresponds to a region of FoxD3 expression (FIG. 18M).

Dissection of NC1

To reduce the size of NC1 (SEQ ID NO: 4) and NC2 (SEQ ID NO: 5) to theminimal core regions, primers (Table 1) were designed to remove portionsfrom the ends of the enhancers. The conservation of the enhancers wasused as a guide to identify the most important regulatory regions. Usingthis approach, NC1 (SEQ ID NO: 4) was reduced to a 550 bp fragmentreferred to as NC1.1 (SEQ ID NO: 7) (FIG. 19) and does not show a lossof activity (FIGS. 20A, 20B). A further deletion to a 300 bp fragmentreferred to as NC1.2 (SEQ ID NO: 8) (FIG. 19) resulted in weak EGFPexpression specifically in the cranial neural crest (FIGS. 20C, 20D),suggesting that the regions at the ends of the 300 bp fragment enhanceactivity of the enhancer, however the critical regions are presentwithin the 300 bp fragment. The core 300 bp NC1.2 fragment (SEQ ID NO:8) was further analyzed by substituting 100 bp regions of sequencewithin the larger 550 bp fragment. This analysis revealed that 200 bpwas required for expression of the enhancer. 20 bp blocks weresubstituted across this region, and a region of 80 bp was found that wascritical for detectable expression of EGFP shown in Mut.7, Mut.8 (FIGS.20E-20H). A further 80 bp region was required as a unit for EGFPexpression, however the individual 20 bp regions within this secondaryregion when substituted only resulted in weakened EGFP expression. (Mut.12, FIGS. 20I, 20J). None of the substitutions resulted in expansion ofthe enhancer-driven expression. The 172 bp fragment (NC1.3) (SEQ ID NO:9) (FIG. 19) containing the most critical and supportive regions wasamplified and electroporated into embryos, and the 80 bp putative coreregion (NC1.4) (SEQ ID NO: 10) was tested in tandem by placing twocopies into the ptkEGFP construct (see Example X). The 172 bp fragment(SEQ ID NO: 9) alone drove very weak expression of EGFP in the neuralcrest. Two copies of the 80 bp core region (SEQ ID NO: 10) wassufficient to drive EGFP expression in the same pattern as thefull-length NC1 enhancer, albeit slightly weaker, suggesting that the 80bp region contains the core elements essential for activity of thisenhancer.

Binding site mutations were made within NC1. Potential transcriptionfactor binding sites within the core region were identified usingRvista, MatInspector and Jasper (FIG. 21A). Mutations were made to thesesites by substituting 6-8 bp of the core binding site as shown in Table2. Mutations to the Ikaros binding site or to the Ets/Zeb binding sitedid not affect expression of EGFP (FIGS. 21B, 21C), however mutation ofthe homeodomain site or Ets/Gata site resulted in loss of EGFPexpression (FIGS. 21D-21G). Msx1 and Lmx1b are two of the homeodomainproteins expressed in the neural folds prior to expression of FoxD3, andcandidates for regulation of FoxD3. Ets1 is expressed specifically inthe cranial neural crest just after the onset of FoxD3 expression. Wetested whether these genes could be regulating FoxD3 expression byco-electroporating one or more FITC-conjugated morpholinos to knockdownthese genes together with an NC1 reporter directing expression ofCherry. There was no effect on the expression of the NC1 enhancer or onendogenous FoxD3 using a morpholino against Ets1 (at 1 or 2 mM).

Dissection of NC2

The NC2 (SEQ ID NO: 5) region was analyzed using the same process ofdeletions and substitutions as that employed to dissect NC2 (FIG. 22).Interestingly, the most highly conserved region of NC2 was not requiredfor activity of the enhancer, and when tested alone drove no activity.This allowed reduction of the full-length enhancer to a 1339 bp fragmentcalled NC2.3 (SEQ ID NO: 11) (FIG. 22), in which all further analysiswas conducted. NC2.4 (SEQ ID NO: 12) is a 600 bp fragment that directsexpression in the vagal and trunk neural crest, but not in the cranialmigrating crest. 100 bp and 30 bp substitutions within NC2.9 (FIG. 22),isolated from NC2.3 narrowed the required regions of the enhanceractivity in the vagal and trunk neural crest to approximately 120 bp,with auxiliary regions on either side of the core being required forstrong expression.

FIG. 23 shows a chart summarizing the EGFP expression when “driven” byvarious NC1 and NC2 subfragments and mutations as indicated.

As shown in FIGS. 17-22, NC1, NC2 and SC1 regulate stages of FoxD3expression. In one embodiment, NC1, NC1.1, NC1.2, NC1.3 or NC1.4 (SEQ IDNOS: 4, 7-10) is used as a vector expression “driver” to identify cellsor manipulate in vivo expression (e.g. gene knockdowns using miRNA,morpholinos, etc) at stages HH8-HH14. In another embodiment, NC2 (SEQ IDNO: 5) is used as a vector expression “driver” to identify cells ormanipulate in vivo expression at stages HH9-HH24. In another embodiment,SC1 (SEQ ID NO: 6) is used as a vector expression “driver” to identifycells or manipulate in vivo expression at stages HH8-HH24. Cells or invivo expression can be identified or monitored using a number of methodsknown in the art—e.g. the “driven” or activated expression can be of anyprotein that can be monitored. As shown herein, the EGFP and RFPproteins are expressed and the fluorescence is imaged. The enhancersequences can “drive” the expression of any protein that can bemonitored via specific antibody to the protein, or an antibody to a tagfused with the protein. Fluorescent expression allows for theutilization of flow cytometry to specifically isolate and sort thosecells which are able to activate the particular enhancer region, fromthose which cannot.

Binding Sites in the Enhancer Regions of SEQ ID NOS 1-12

The presence of transcription factors and their ability to bind a DNAsite is the mechanism for activating an enhancer region that thensubsequently activates expression of an endogenous gene or expressionprotein in a vector construct. Specific manipulation of the bindingsites, which are commonly 6 basepairs in length, indicates that a onebase pair mutation within the six, does not disrupt transcription factorbinding. However, two or more mutations within 6 basepairs, (in atranscription factor binding site), has shown disrupted reporterexpression, due to the inability for the transcription factor to bind.Accordingly, in one embodiment, an enhancer region is provided having aDNA sequence of any one of SEQ ID NOS: 1-12 or a DNA sequence of any oneof SEQ ID NOS: 1-12 having one or more nucleotide mutations, but no morethan one mutation within any 6 consecutive nucleotides. More than onenucleotide mutation in a region outside of a transcription factorbinding site does not disrupt reporter expression.

As mentioned previously, early naive cells contain regulatory factorscharacteristic of multipotent tissue and are, thus, competent to switchon a neural crest-like transcriptional program in response to the propersignal inputs. Accordingly, in one embodiment, an enhancer region of thepresent invention (any one of SEQ ID NOS: 1-12) is utilized in earlyuncommitted cells to induce neural crest traits. The early uncommittedcells are any cells sharing similar neural crest develop, e.g. human,mouse, rat, avian.

Short-hairpin RNA

The critical issue with short-hairpin vectors is that their constitutiveexpression driving high copy numbers within a cell which, followingsplicing, results in large numbers of MiR vector arms saturating thecell transcriptional machinery. To control this, in one embodiment, ashort-hairpin miRNA system is disclosed wherein short hairpins aregenerated at lower, but still effective levels and whose expression islimited to a specific time and location within the developing embryo.This offers the further advantage of addressing more specific questionson the role of a particular gene at a particular time or location duringdevelopment.

In FIGS. 24A, 24B two alternative vectors are shown. The vector of FIG.24A provides a strong knockdown of (down-regulation of) gene expressionwith moderate EGFP expression, and the miRNA vector of FIG. 24B which isused in parallel with the EGFP as a tracer, gives a strong EFGP signalbut with weaker knockdown. The example vector shown here combines theNC1 (SEQ ID NO: 4) enhancer driving expression of a short hairpin RNAdesigned against Sox10 (‘FoxD3-shSox10’) or against RFP (‘FoxD3-shRFP’)for control. This is expressed in the pre-migratory cranial neural crestfrom approximately 4 somite stage to the 10 somite stage. The EGFPexpression (FIGS. 27A, 27B) and mRNA expression patterns observed within situ hybridization for each vector (FIGS. 26A, 26B). The sameknockdown was analysed by QPCR (FIG. 26C). The loss of Sox10 expressionon the targeted side is apparent in shSox10 vectors but not in shRFPcontrol vectors. FIG. 26D shows in situ hybridization of Sox10 mRNA.

Proof of specificity is the major test for any method of gene knockdown.To demonstrate that no non-targeted genes are affected by the expressionof the vector, RNA was extracted from control and electroporated sidesof embryos expressing either FoxD3(NC1)-shSox10 or FoxD3 sh-RFP.Following cDNA synthesis, QPCR was performed for a panel of 3 relatedneural crest specifier genes—FoxD3, Sox9 and MSX. Sox9 and MSX levelsare unaffected by introduction of either vector, demonstrating that theloss of Sox10 was not due to uncontrolled side effects within the earlyembryo. The loss of FoxD3 is caused by feedback and cross-regulationwith Sox10, indicating that Sox10 is necessary for the maintainedexpression of FoxD3 within the embryo.

An analysis comparing the use of existing pRFPRNAi vectors (Das et al2006) to morpholinos was performed by (Mende et al 2008) who observethat sh-MiR vectors cause many non-specific defects and recommendagainst using these in early embryos. These non-specific effects includeboth ectopic and loss of expression of non-targeted genes. It was foundthat, irrespective of the gene targeted, these vectors disrupted oticcup morphology and caused loss of otic markers such as Pax2. As afurther control for specificity, we performed in situ hybridization onembryos expressing the FoxD3premigratory-shSox10 vector. We find thatotic cup morphology and Pax2 expression is unaffected in theelectroporated half compared to the non-electroporated control.

Although short hairpin based RNA interference vectors exist for avianmodel systems (Das et al. 2006) their use in early development has beenlimited by non-specific effects resulting from constitutive expressionand high expression levels. These compromise the accuracy of ourunderstanding of gene function gained by studying the knockdown. Forthese reasons, a vector system where targeted knockdown can becontrolled to endogenous levels and locations using enhancer activity isadvantageous. This offers a significant improvement on existing vectors.

Isolating Neural Crest Cells Using Neural Crest Enhancer DNA Fragments

In one embodiment, a method for isolating a specific Hamburger andHamilton stage or stages of neural crest cells using enhancer DNA of thepresent invention as the driver for EGFP expression in a vector. Afterelectroporation of the enhancer activated EGFP vector into the chickembryos, embryonic neural crest cells having the stage-specific enhancerbinding proteins will activate the EGFP, and these cells can be isolatedby flow cytometry as further described in the Examples.

Infection of Sox10 and FoxD3 Enhancers into Human Embryonic Stem (ES)Cells

Sox10E (SEQ ID NO: 1) and NC1 (SEQ ID NO: 4) were both separatelysubcloned into an RFP lentiviral vector. Human embryonic stem (ES) cellswere infected with each vector separately, differentiated into neuralcrest fate and imaged by microscopy for RFP expression. Both NC1 (FIG.28A) and Sox10E (FIG. 28B) were activated to direct RFP expression inthose cells. The enhancers of the present invention function in humancells. Given the stage-specificity of the enhancers as disclosed herein,an enhancer construct of the present invention subcloned into humancells would allow for identification and isolation of cellscorresponding to the stage(s) represented by the enhancer region. In oneembodiment, enhancers of the present invention are used to identifyearly stage cells. In another embodiment, enhancers of the presentinvention are used to identify early stage human cells. In anotherembodiment, enhancers of the present invention are used to induce neuralcrest traits with directed differentiation in early stage uncommittedcells.

Conservation and Function of Homologous Sequences Across Species

As discussed and shown herein, the Sox10 and FoxD3 enhancers and theirsubfragment sequences (SEQ ID NOS: 1-12) are conserved across severalspecies. It has been shown previously that enhancer sequences from onespecies can replace endogenous enhancers of another species, as reportedin Abbasi et al., 2007, PLoS ONE, 2(4): e366. Homologous sequences areknown to function in vivo across species as shown herein (FIGS. 28A,28B) and as reported in Jiang et al. 2009, Stem Cells Dev. 18(7):1059-1070). Thus, the chick embryonic enhancer sequences as describedherein are not limited to in vivo function in the chick embryo, and canbe utilized in other organisms, including, but not limited to, human,mouse, rat, opossum, zebrafish and xenopus.

Accordingly, in one embodiment, an isolated DNA sequence is provided,wherein the sequence has at least 60% homology to one selected from thegroup consisting of SEQ ID NOS: 1-12. In another embodiment, an isolatedDNA sequence is provided, wherein the sequence has at least 70% homologyto one selected from the group consisting of SEQ ID NOS: 1-12. Inanother embodiment, an isolated DNA sequence is provided, wherein thesequence has at least 80% homology to one selected from the groupconsisting of SEQ ID NOS: 1-12. In another embodiment, an isolated DNAsequence is provided, wherein the sequence has at least 90% homology toone selected from the group consisting of SEQ ID NOS: 1-12. In anotherembodiment, an isolated DNA sequence is provided, wherein the sequencehas at least 95% homology to one selected from the group consisting ofSEQ ID NOS: 1-12

EXAMPLES/METHODS

Comparative genomic analyses and cloning of Sox10 and FoxD3 regulatoryregions. Highly conserved genomic regions were identified using ECRbrowser. Binding motifs were predicted using Jaspar database and P-Matchprogram from Transfac database. FIGS. 1 and 17 show a schematic diagramshowing comparative genomic analysis using ECR browser.

In FIG. 1, chicken, zebrafish, Xenopus, opossum, mouse, rat and humangenomic sequences were compared between Sox10 and neighboring genes,Slc16A8 and PolR2F. Red peaks=highly conserved elements; blue=codingexons; green=transposable elements and simple repeats. Boxed Sox10putative regulatory regions L8(L=late) and E(E=early) show activity inneural crest. UTRs shaded in yellow.

FIG. 17 shows the genomic region of chicken FoxD3 compared to othervertebrates using the UCSC and Rvista programs. The genomic regionanalyzed was 160 kb, from the 3′ end of the Atg4C gene immediatelyupstream of FoxD3, to the start of the Alg6 gene immediately downstreamRegions that were conserved across most or all vertebrates wereidentified and primers were designed to amplify the conserved regionsfrom BACs CH261-166E22 and CH261-100C15. The primers used to amplifythese regions and further dissect these regions, are shown in Table Y.The amplified regions were directionally cloned into the ptkEGFP vector(Uchikawa et al., 2003) donated by H. Kondoh, Osaka University, Japan)using KpnI and XhoI sites.

In general, putative regulatory regions were amplified with Expand HighFidelity Plus (Roche, Indianapolis, Ind.), from chicken BAC DNA (BACPAC,Oakland, Calif.) and cloned into the ptk-EGFP vector (Uchikawa et al.(2003) Dev Cell 4(4):509-519). Sox10 and FoxD3 genomic regions wereamplified with the Expand High Fidelity Plus PCR System using BAC DNAclones as the template (Chicken BAC library Chori 26). Each fragment,ranging from ˜3 kb-5 kb in size, was cloned into the SmaI-linearizedptk-EGFP vector. The ptk-EGFP reporter vector has the Herpes simplexvirus thymidine kinase basic promoter upstream of enhanced GFP and was akind gift of Dr. Hisato Kondoh. The clones with the appropriateorientation were identified by colony PCR and sequenced. The plasmid DNAof the correct clones was prepared and purified using the Endo-free maxikit (Qiagen) and eluted in EDTA-free buffer.

ptk-Cherry and pCI H2B-RFP plasmids were generated for use in thisstudy. ptk-Cherry reporter vector was made by swapping EGFP with Cherryfluorescent protein in the ptk-EGFP reporter vector (Uchikawa et al.(2003) Dev Cell 4(4):509-519). pCI H2B-RFP, a tracer construct thatyields ubiquitous expression under the control of chicken beta actinpromoter is a bicistronic vector allowing for exogenous expression ofproteins of interest and of a fusion protein of human histone 2B andmonomeric RFP protein, translated from the IRES. The pCI H2B-RFPconstruct was made by replacing the 3×NLS-EGFP sequence within thepCI-GFP vector (Megason S G & McMahon A P (2002) Development129(9):2087-2098) with the H2B-mRFP1 sequence.

In situ hybridization. Whole-mount in situ hybridization was performedusing a procedure previously described (Wilkinson D G (1992) In situHybridization: A Practical Approach, ed Wilkinson DG (IRL Press,Oxford), pp 75-83). Whole mounts were imaged using microscope andAxiovision camera and software. Fluorescent in situ procedure using GFPprobe was adapted from (Acloque and Nieto (2008) Methods Cell Biol87:169-185). Whole-mount in situ hybridizations were performed using aprocedure previously described (Antonellis A. et al. (2008) PLoS Genet4(9):e 1000174). Fluorescent in situ procedure using GFP probe wasadapted from (Dutton et al. (2008) BMC Dev Biol 8:105. With theexception of the Sox9 and Sox10 probes, which were prepared using fulllength cDNA constructs (a gift from Yi-Chuan Cheng) (Cheng et al. (2000)Brain Res Dev Brain Res 121(2): 233-241), as a template, all otherdigoxigenin-labeled antisense RNA probes, were prepared from chicken ESTclones obtained from (ARK Genomics and MRC geneservice). Sox10 templatewas digested with HindIII, while all EST clones were linearized usingNotI restriction enzyme. All antisense RNA probes were synthesized usingT3 RN A polymerase, according to standard protocols. FIG. 3B shows thatat HH8+, GFP transcripts are detected by fluorescent in situhybridization in cranial neural crest (CNC) similar to endogenous Sox10expression (FIG. 3G). Distribution of EGFP transcripts (FIGS. 3C, 3D,3E; HH9+; HH12, H15, respectively) is similar to endogenous Sox10 inFIGS. 3H-3J, respectively. FIG. 3D shows EGFP expression at HH12 inrhombomere5 stream surrounding the otic vesicle(OV) resembles endogenousSox10 (FIG. 3I), but is missing in vagal neural crest (VNC). (FIG. 3F).Cross section of embryo in FIG. 3D shows specific Sox10E regulatoryactivity in CNC around optic vesicle(OpV). (FIGS. 3G-3J) show endogenousSox10 expression at HH8+−HH15.; (OP is otic placode).

Ex ovo and in ovo electroporations. Chicken embryos were electroporatedat stages HH4-HH8 to target the cranial neural crest cell population andat stages HH10-12 to target vagal and/or trunk neural crest cellsfollowing previously described electroporation procedures(Sauka-Spengler and Barembaum (2008), Methods Cell Biol 87:237-256). Inex-ovo experiments, the DNA plasmid constructs (enhancer driven reporterwith ubiquitously-expressing tracer) were introduced in the entireepiblast of the early chicken embryo, while in in-ovo electroporationsonly one half of the neural tube received the DNA. Injected DNA plasmidconcentrations were as follows: 2 μg/μl of ptk-EGFP or ptk-Cherryreporter construct, containing each of Sox10 or FoxD3 putativecis-regulatory regions or the Sox10E2, NC1, NC2 and SC1 mutatedversions, combined with 1 μg/μl of either tracer (pCI H2B-RFP) orexpression constructs (Sox9-pCI H2B-RFP or Ets1-pCI H2B-RFP).

Microscopy and immunohistochemistry. The electroporated embryos werecollected at different stages, fixed in 4% paraformaldehyde O/N and thenwashed three times in PBS at room temperature. A Zeiss axioskop2 Plusfluorescence microscope equipped with the AxioVision software wasemployed to image the embryos. Images were processed using AdobePhotoshop CS2. After imaging, embryos were cryo-protected in two steps:15% sucrose/PBS and 7.5% gelatin/15% sucrose/PBS, equilibrated andmounted in 20% gelatin/PBS and frozen in liquid nitrogen. 12 μmcryosections were collected on Super Frost Plus slides (FischerScientific, Pittsburgh, Pa.) and de-gelatinized for 2×10 minutes at 42°C. in PBS. To intensify EGFP signal, the sections were washed 4× in PBSfor 5 minutes, blocked for 1 hour in 10% Donkey serum/PBTW (PBS/0.1%Tween-20) and stained with 1:1000 anti-GFP primary antibody (Abeam Inc.,Cambridge, Mass.) followed by 1:2000 Donkey anti-goat Alexa-Fluor488-conjugated secondary antibody (Molecular probes). Sections weresubsequently washed, cover slipped and imaged using the same imagingprocedure described for the in situ whole-mounts.

Dissection of Sox10 downstream putative regulatory region and mutationof candidate binding sites. The 3.5 kb genomic region downstream Sox10coding (Sox10E) was divided into smaller regions, dissected and mutatedfragments amplified using Expand High Fidelity Plus PCR System (RocheApplied Science, Indianapolis, Ind.). For the initial dissection of theSox10E fragment, the following primers were used:

Sox10E1_5′, (SEQ ID NO: 19) 5′-ATTAGGTACCTCTGATACAGATGCAAGGCTG-3′Sox10E1_3′, (SEQ ID NO: 20) 5′-TAATCTCGAGAATTTGCAGCACTGTGGCCTT-3′;Sox10E2_5′, (SEQ ID NO: 21) 5′-AATTGGTACCGGCAAGAGTGGCAATTTAACC-3′Sox10E2_3′, (SEQ ID NO: 22) 5′-ATTACTCGAGATTGCTTCCCCCTAGACAGTT-3′;Sox10E3_5′, (SEQ ID NO: 23) 5′-TTTTGGTACCTAACCAGGGAGGAGTTGTGG-3′Sox10E3_3′, (SEQ ID NO: 24) 5′-AATTCTCGAGAAGGCCCACAGCAGAGTG-3′.

To perturb candidate binding sites within the Sox10E2 fragment, we usedsingle or fusion PCR as previously described (Nikitina N, Sauka-SpenglerT, & Bronner-Fraser M (2008) Proc Natl Acad Sci USA 105(51):20083-20088;Sauka-Spengler T & Bronner-Fraser M (2006) Curr Opin Genet Dev16(4):360-366).

The primers having mutations are listed here. Mutated regions areunderlined, the mutated nucleotides are shown in bold and fusion primersequences are italicized:

First 2 Pax sites clusters (M1, M3)_5′, (SEQ ID NO: 25)5′-AATTGGTACCGGCAA AGCCCATG -ATTTAACCTACAACTGCTGAGCTTGTAGGA AGCCCATGGGCGACTGTGCTTCCGGCT-3′; Myb (M2)_5′, (SEQ ID NO: 26)5′-ATTAGGTACCTGGCAAGAGTGGCAA GGGATGGACTGGTAGATGGAAGTGTAGGACTGTGACTGGCGA-3′; Second 2 Pax cluster sites (M5, M7)_3′,(SEQ ID NO: 27) 5′-TCCCTGCTCCTGCTGCTTATCA TGGGCT GGGATCCCCTTTCA TGGGCTCTGCCCCAGCCGGAAGCACAGT-3′; Ets/Elk (M4)_5′, (SEQ ID NO: 28)5′-ATTAGGTACCTGGCAAGAGTGGCAATTTAACCTACAACTGCTGAGCTTGTAGGACTGTGACTGGCGACTGTATGGTTAAT TGGGGCAGTGCCACTGAAA-3′; NFKB1 (M6)_3′,  (SEQ ID NO: 29)5′-TGCTGCTTATCAGTGATG AGCCCATGGTCTCAGTGGCACTGCCCCAG3′;Lef/Tcf/SoxE (M8)_3′, (SEQ ID NO: 30) 5′-TCTCATCAAATCACCT CCATCTACCCTGCTCCTGCTGCTTATCAGT-3′; Ets (M9) 3′, (SEQ ID NO: 31)5′-AATTCTCGAGATTGCTTCCCCCTAGACAGTTGGGCCTTTGTGCCCTGAGCAGGTTGCTGTGGAAACCCCCAATGGG CTCTCTGGCCAGAGCTGGCT-3′; NFKB1/Lef/Tcf/Ets1_3′, (SEQ ID NO: 32)5′-AATTCTCGAGTTGCTTCCCCCTAGACAGTTGGGCCTTTGTGCCCTGAGCAGGTTGCTGTGGAGCCCATGGTC TTCCTCTCTGGCCAGAGC-3′; SoxE/Lef/Tcf (M10)_3′,(SEQ ID NO: 33) 5′-ATTACTCGAGATTGCTTCCCCCTAGACAGTTGGGC G T A TG CGCCCTGAGCAGGTTGCTGTGGAAA-3′; Myb (M11)_3′, (SEQ ID NO: 34)5′-ATTACTCGAGATTGCTTCCCCCTA CTCCATAA GGCCTTTGTGCCCTGAGCA-3′;SoxD (M12)_3′, (SEQ ID NO: 35) 5′-ATTACTCGAG G CCAATTCCCCCTAGACAGTTGGGC-3′; Δ1_3′, (SEQ ID NO: 37)5′-AATTTCCTCTCTGGCCAGA AAATCACCTATTGTTTCCCT-3′; R1_3′, (SEQ ID NO: 37)5'-AATTTCCTCTCTGGCCAG CCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATCGTCTAAATCACCTATTGTTTCCCT-3′.

After PCR amplification, each fragment was purified using PCR productpurification kit (Qiagen) and cloned into KpnI/XhoI digested ptk-EGFP orptk-Cherry reporter vectors.

Dissection of Sox10E reveals two regulatory regions that function in aspecific spatiotemporal manner: Sox10E2 is activated as cranial neuralcrest cells delaminate whereas Sox10E1 is activated in later migratingvagal and trunk neural crest. As shown in FIG. 4A, Sox10E1 displays noactivity in the delaminating cranial neural crest. FIG. 4B shows Sox10E2activity at HH15 persists in the periocular crest and otic vesicle, butalso within the first two branchial arches, which lack endogenous Sox10;Sox10E2 is not expressed in vagal neural crest at this stage or later,at HH18 shown in FIG. 4C, and in either the vagal or trunk regions(FIGS. 4D-4F). Panels corresponding to (4A-4C), respectively, showexpression of the co-electroporated tracer pCI H2B-RFP to locate cellsthat received both tracer and reporter EGFP plasmid DNA.

Sox10E contains distinct spatiotemporal regulatory elements: Sox10E2 indelaminating CNC and Sox10E1 in later migrating vagal (VNC) and trunkneural crest. FIG. 5A Schematic diagram representing dissection ofSox10E fragment, located ˜1 kb downstream of Sox10 locus (UTR inyellow). Two smaller active regulatory fragments embedded within Sox10E,(Sox10E1&E2) each contain a conserved region(red bar) with 70% sequencehomology between amniotes. (FIG. 5B) Sox10E2 drives expression indelaminating CNC(arrows) at HH8+; Sox10E11 is first active in migratingVNC at HH15 (FIG. 5C). (FIG. 5D) Sox10E1 activity persists in migratingVNC, trunk neural crest (arrow), and branchial arches 3-5(arrowhead).(FIG. 5E) Table 11 summarizes distinct temporal (HH9-18) and spatial(cranial/vagal/trunk) regulatory activity of Sox10E1&E2. Red−=noexpression; green+=EGFP reporter expression.

Dissection of the Sox10E2 fragment reveals an essential core andauxiliary region important for optimal enhancer activity. FIG. 6A showsa magnified image showing the region Sox10E2 from the genomiccomparative analysis illustrated in FIG. 4A. Each uncolored peakrepresents the conserved region equivalent to region Sox10E2 in chickenfor each corresponding species. Inside the conserved regions, the redpatches represent highly conserve portions (85%-90% each 30 bp). Theorder of species from top to bottom: mouse, dog, opossum, rat,chimpanzee and human. FIG. 6B shows a schematic diagram representing thedifferent successive deletions (dotted lines) that were performed,guided by bioinformatics, to identify the main core element responsiblefor the regulatory activity of the Sox10E2 fragment. The horizontalblack lines represent different fragments. Red portion of the linesdenote a 160 bp region (referred to as essential core element), highlyconserved between dog, chimpanzee and human, capable of producing weaktissue-specific regulatory activity in delaminating cranial neural crestcells. The gray arrow points to a non-conserved 59 bp-long auxiliaryregion, necessary for achieving strong regulatory activity of theenhancer. CNC, cranial neural crest.

Transcriptional inputs into the Sox10E2 regulatory region. FIG. 7A showsa schematic diagram showing sequence alignment of 264 bp Sox10E2regulatory region; essential core region shaded in yellow. Coloredframes indicate computationally identified putative transcriptionfactors binding motifs. Mutations M1-M13 were replaced by randomsequences. Faded sequence shows a 45 bp region deleted or replaced bymCherry coding sequence. Highlighted in blue are conserved nucleotideswithin putative binding motifs. Single dashed lines indicate no bases inaligned sequence. Thick dashed lines indicate nonalignable sequences.Thick solid underlines delineate Sox10E2 subfragments used in EMSA andpulldown assays. Sox10E2-driven EGFP expression in CNC (FIG. 7B) isabolished upon mutation of an Ets1 binding motif (FIG. 7C), but onlydecreased after mutation of putative SoxD motif (FIG. 7D), and notaffected by simultaneous mutation of four putative Pax sites(FIG. 7E).Simultaneous inactivation of SoxE, Ets and Myb binding sites(M2,M8,M9,M11,M12) within a much larger genomic region abolishesreporter expression in delaminating CNC (FIG. 7F).

Dissection of FoxD3 NC1. NC2 and SC1 Regions

Enhancer substitutions and mutations: regions of NC1 and NC2 werereplaced with EGFP coding sequence using fusion PCR. For 100 bpsubstitutions, the region of EGFP used was (SEQ ID NO:38)(tggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaactt-caagatccgccacaacatcgaggacgg):for 20 bp substitutions (SEQ ID NO: 39)(tggagtacaactacaacagc), and for30 bp substitutions (SEQ ID NO: 40) (acaagcagaagaacggcatcaaggtgaact).Amplified fusion constructs were cloned into ptk-EGFP vector (Uchikawaet al., 2003) and sequenced to ensure no additional mutations werepresent. Binding sites were identified using Jasper. RVista andMatInspector databases and programs. Sites were mutated by substituting6-8 adjacent critical base pairs with GFP coding sequence, using fusionPCR and cloning into ptkEGFP. Mutated enhancer constructs wereelectroporated into stage 4 embryos as described herein and analyzed forexpression of EGFP and RFP at stages 8-12.

TABLE 1 Text in capitals indicates enhancer sequence, and text in small lettersindicates replacement GFP sequence. To make the mutated constructs, mutatedprimers were paired with flanking primers (NC1.1 or NC2.9), amplified and joinedin a fusion PCR reaction using the flanking primers. Primer NamePrimer Sequence NC1 fwd (SEQ ID NO: 41) AGGCAATGCAGCGAATGACC NC1 rev(SEQ ID NO: 42) GACCTGGCTCCCTTTGAGAC NC1.1 fwd (SEQ ID NO: 43)CAGTAAGCTTTCCACCAACA NC1.1 rev (SEQ ID NO: 44) GTTTAACATACACTATCCAATGNC1.2 fwd (SEQ ID NO: 45) CCTGAAGCTCATTAGATATT NC1.2 rev (SEQ ID NO: 46)CCGACATTTGGGAAATTAAA NC1.3 fwd (SEQ ID NO: 47) CATTAGATATTCCCTGGNC1.3 rev (SEQ ID NO: 48) AAAGAGGCCAGTAATTGTC NC1 80 bp core fwd(SEQ ID NO: 49) CATTAGATATTCCCT NC1 80 bp core rev (SEQ ID NO: 50)CCCACAGAGATTGCT NC1.1 Mut 1 fwd  (SEQ ID NO: 51)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggGAGACTCTCCTNC1.1 Mut 1 rev (SEQ ID NO: 52)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaAAACCCACTGAA NC1.1 Mut 2 fwd (SEQ ID NO: 53)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggACTTAAGCTAAATGG NC1.1 Mut 2 rev (SEQ ID NO: 54)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaCTCCTATTGGCA NC1.1 Mut 3 fwd (SEQ ID NO: 55)caagcagaagaacgg catcaaggtgaacttcaagatccgccacaa catcgaggacggCGGATTTTGGTGNC1.1 Mut 3 rev (SEQ ID NO: 56)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaCGATTAATGTAACTC NC1.1 Mut 4 fwd (SEQ ID NO: 57)agtacaactacaacagcCATTAGATATTCC NC1.1 Mut 4 rev (SEQ ID NO: 58)tgttgtagttgtactccaAAGAATTCTCTTTT NC1.1 Mut 5 fwd (SEQ ID NO: 59)agtacaactacaacagcATTCTCCTAAGTA NC1.1 Mut 5 rev (SEQ ID NO: 60)tgttgtagttgtactccaAGCTTCAGGAAA NC1.1 Mut 6 fwd (SEQ ID NO: 61)agtacaactacaacagcTAACAGGATTTTC NC1.1 Mut 6 rev (SEQ ID NO: 62)tgttgtagttgtactccaATAGGCCAGGGA NC1.1 Mut 7 fwd (SEQ ID NO: 63)agtacaactacaacagcGAGCAATCTCTG NC1.1 Mut 7 rev (SEQ ID NO: 64)tgttgtagttgtactccaTAAAATCTAATTACTTAG NC1.1 Mut 8 fwd (SEQ ID NO: 65)agtacaactacaacagcAATAGGAGACTC NC1.1 Mut 8 rev (SEQ ID NO: 66)tgttgtagttgtactccaTGATCTGTTGAAA NC1.1 Mut 9 fwd (SEQ ID NO: 67)agtacaactacaacagcTCTGGCCTTACC NC1.1 Mut 9 rev (SEQ ID NO: 68)tgttgtagttgtactccaCCCACAGAGATT NC1.1 Mut10 fwd (SEQ ID NO: 69)agtacaactacaacagcAGCATGGATAAC NC1.1 Mut 10 rev (SEQ ID NO: 70)tgttgtagttgtactccaGGGAGAGTCTCCTA NC1.1 Mut 11 fwd (SEQ ID NO: 71)agtacaactacaacagcTGGGAGTTAATAG NC1.1 Mut 11 rev (SEQ ID NO: 72)tgttgtagttgtactccaGCCCTGCTGGTAA NC1.1 Mut 12 fwd (SEQ ID NO: 73)agtacaactacaacagcACTGGCCTCTTT NC1.1 Mut 12 rev (SEQ ID NO: 74)tgttgtagttgtactccaGCCTGGATGTTA NC1.1 Mut 13 fwd (SEQ ID NO: 75)agtacaactacaacagcTTACATTAATGCACT NC1.1 Mut 13 rev (SEQ ID NO: 76)tgttgtagttgtactccaAATTGTCCTATTAAC NC1.1 Mut 14 fwd (SEQ ID NO: 77)agtacaactacaacagcACTTAAGCTAAATG NC1.1 Mut 14 rev (SEQ ID NO: 78)tgttgtagttgtactccaAAAGAGGCCAGTA NC2 fwd (SEQ ID NO: 79)TGAGTGTGCCTCCATGTGTC NC2 rev (SEQ ID NO: 80) GATGGTGCAGCACACGGTTGNC2.1/NC2.4 rev (SEQ ID NO: 81) TGTGGTAGGCTTATTGTTTTGCT NC2.2 rev(SEQ ID NO: 82) TCGGTTTTGTTTCACAGTTTG NC2.3/NC2.4 (SEQ ID NO: 83)GGTGCATAGAACAAACTGTG NC2.6/NC2.9 fwd NC2.5 fwd (SEQ ID NO: 84)GCACTGGGTTCATGAAGTTTC NC2.5 rev (SEQ ID NO: 85) CTACCTCAGAAGGCATTGTANC2.7/NC2.8 fwd (SEQ ID NO: 86) CGATTCTCTGTCTGCCAATTT NC2.8/NC2.9/(SEQ ID NO: 87) GTTCACCCAGTAAACCAGTA NC2.10 rev NC2.10 fwd(SEQ ID NO: 88) TGTCATCTTCCGCTCACTT NC2.9 Mut 1 fwd (SEQ ID NO: 89)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggGGAAACGATGGNC2.9 Mut 1 rev (SEQ ID NO: 90)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaGACATAACTTTGTC NC2.9 Mut 2 fwd (SEQ ID NO: 91)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggAAATTACTCCGATTNC2.9 Mut 2 rev (SEQ ID NO: 92)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaCCAAATACTTTCACT NC2.9 Mut 3 fwd (SEQ ID NO: 93)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggTAGCAAGGGGCTTNC2.9 Mut 3 rev (SEQ ID NO: 94)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaGTATCATTTCAATTAG NC2.9 Mut 4 fwd (SEQ ID NO: 95)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggCCGCTACCTTCANC2.9 Mut 4 rev (SEQ ID NO: 96)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaCAGCAGCCACTT NC2.9 Mut 5 fwd (SEQ ID NO: 97)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggGTATTCATCCCCAANC2.9 Mut 5 rev (SEQ ID NO: 98)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaTGGTAGGCTTAT NC2.9 Mut 6 fwd (SEQ ID NO: 99)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggCAGTAGGAAAAACNC2.9 Mut 6 rev (SEQ ID NO: 100)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaAACACTTATCTCTAC NC2.9 Mut 7 fwd (SEQ ID NO: 101)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaacT atcgaggacggTAGTTCAACTGTGNC2.9 Mut 7 rev (SEQ ID NO: 102)gatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccaAACCGAGCGCAA NC2.9 Mut 8 fwd (SEQ ID NO: 103)caagcagaagaacggcatcaaggtgaacttcaagatccgccacaaca tcgaggacggTCAGTGCAATTCNC2.9 Mut 8 rev (SEQ ID NO: 104)gatgccgttcttctgcttgtcggccatgatatgacgttgtggctgttgtagttgtactccaGAACTATTTTGGAT NC2.9 Mut 9 fwd (SEQ ID NO: l05)gaagaacggcatcaaggtgaactATGGGCAATAAT NC2.9 Mut 9 rev (SEQ ID NO: 106)ttgatgccgttcttctgcttgtCCAAATACTTTCA NC2.9 Mut 10 fwd (SEQ ID NO: 107)gaagaacggcatcaaggtgaactACCTCCCTGTTA NC2.9 Mut 10 rev (SEQ ID NO: 108)ttgatgccgttcttctgcttgtGGCAACCAAACT NC2.9 Mut 11 fwd (SEQ ID NO: 109)gaagaacggcatcaaggtgaactGAAATGATACAAAT NC2.9 Mut 11 rev (SEQ ID NO: 110)ttgatgccgttcttctgcttgtCATGACTTTTTTG NC2.9 Mut 12 fwd (SEQ ID NO: 111)gaagaacggcatcaaggtgaactCTGCCAATTTAG NC2.9 Mut 12 rev (SEQ ID NO: 112)ttgatgccgttcttctgcttgtAATTAGTTACTAGC NC2.9 Mut 13 fwd (SEQ ID NO: 113)gaagaacggcatcaaggtgaactGGTCAAATGAGC NC2.9 Mut 13 rev (SEQ ID NO: 114)ttgatgccgttcttctgcttgtACAGAGAATCGG NC2.9 Mut 14 fwd (SEQ ID NO: 115)gaagaacggcatcaaggtgaactGTTTGAAGTGGC NC2.9 Mut 14 rev (SEQ ID NO: 116)ttgatgccgttcttctgcttgtCTTGACCACAAC NC2.9 Mut 15 fwd (SEQ ID NO: 117)gaagaacggcatcaaggtgaactCTTGGTGTGGAC NC2.9 Mut 15 rev (SEQ ID NO: 118)ttgatgccgttcttctgcttgtATAGTTTCATGAAT NC2.9 Mut 16 fwd (SEQ ID NO: 119)gaagaacggcatcaaggtgaactCATTACCCCATA NC2.9 Mut 16 rev (SEQ ID NO: 120)ttgatgccgttcttctgcttgtCCCCTTGCTATG NC2.9 Mut 17 fwd (SEQ ID NO: 121)gaagaacggcatcaaggtgaactCACAGATAGCAA NC2.9 Mut 17 rev (SEQ ID NO: 122)ttgatgccgttcttctgcttgtTCCAGAGGAGTC NC2.9 Mut 18 fwd (SEQ ID NO: 123)gaagaacggcatcaaggtgaactGCTACCTTCAGC NC2.9 Mut 18 rev (SEQ ID NO: 124)ttgatgccgttcttctgcttgtCAGCACTCTCCT NC2.9 Mut 19 fwd (SEQ ID NO: 125)gaagaacggcatcaaggtgaactTCTGTGTCAGTC NC2.9 Mut 19 rev (SEQ ID NO: 126)ttgatgccgttcttctgcttgtGGTGGTAGGCTT NC2.9 Mut 20 fwd (SEQ ID NO: 127)gaagaacggcatcaaggtgaactCTGACCAGGATA NC2.9 Mut 20 rev (SEQ ID NO: 128)ttgatgccgttcttctgcttgtGAAGCTTTTGATG NC2.9 Mut 21 fwd (SEQ ID NO: 129)gaagaacggatcaaggtgaactTAAGTGTTGTATTC NC2.9 Mut 21 rev (SEQ ID NO: 130)ttgatgccgttcttctgcttgtAGGCAGCCACTG SC1 fwd (SEQ ID NO: 131)GACAAAGATAACCATCCTCC SC1 rev (SEQ ID NO: 132) CACTTCTTCAATTGCTGAGGSC1.1 rev (SEQ ID NO: 133) ATAGCAAAGACACATTGTGC SC1.1a rev(SEQ ID NO: 134) GTGCAGAAATCAACAGCTA SC1.1b fwd (SEQ ID NO: 135)GGCCATTATGATCTTTAACT

TABLE 2  Primer name Primer sequenceNC1.1 Ikaros mut. Fwd (SEQ ID NO: 136) gaCTACAAGAGCcctattctcctaNC1.1 Ikaros mut. rev (SEQ ID NO: 137) aggGCTCTTGTAGtctaatgagcttNC1.1 Ets/Zeb mut. fwd (SEQ ID NO: 138) ctattTAGAACagtaattagattttaNC1.1 Ets/Zeb mut. rev (SEQ ID NO: 139) tactGTTCTAaataggccagggaNC1.1 HD mut. fwd (SEQ ID NO: 140) cctaCTACCACCgattttaacaggNC1.1 HD mut. rev (SEQ ID NO: 141) atcGGTGGTAGtaggagaataggNC1.1 Ets/Gata mut. fwd (SEQ ID NO: 142) ttaaACAACAGCtcaacagatcagNC1.1 Ets/Gata mut. rev (SEQ ID NO: 143) tgaGCTGTTGTttaaaatctaattac

The regions that were mutated are indicated by capital letters. Primerswere paired with NC1.1 fwd or rev primers in a first round ofamplification, then joined in fusion PCR using the flanking NC1.1 fwdand rev primers.

EMSA and Pull-down Assays

EMSA was performed using LightShift Chemiluminescent EMSA Kit (ThermoScientific, Rockford, Ill.) following manufacturer's instructions. FiveSox10E2 subfragments (M2, M4, M8, M9, M11/M12) and three controlfragments were obtained by annealing synthetic oligonucleotides with orwithout 5′ biotin modification (IDT Biotechnology). Double strandedfragments used in EMSA assay either had biotin tags on both ends or werenot labeled (cold probes).

The sequences of Sox10E2 subfragments used in these approaches were:

M2, (SEQ ID NO: 144) GCAATTTAACCTACAACTGCTGAGCTTGTA M4, (SEQ ID NO: 145)GGCGACTGTGCTTCCGGCTGGGGCAGTG; M8, (SEQ ID NO: 146)GGAGCAGGGAAACAATAGGTGATTT; M9, (SEQ ID NO: 147)TGGCCAGAGAGGAAATTGGGGGTTT; M11/12, (SEQ ID NO: 148)TCAGGGCACAAAGGCCCAACTGTCTAGGGGG.

The sequences of scrambled controls were selected based on the absenceof binding sites with homology of greater than 70%, according toexhaustive survey of Jaspar and Transfac databases. They were asfollows: Myb Co, (SEQ ID NO: 149) TCTTCAAGTCCGCCAT-GCCCGAAGG; Sox9 Co,(SEQ ID NO: 150) TACGGCAAGCTGTTCATCTGCACCA; Ets1 Co, (SEQ ID NO: 151)ATGTCTACGTCGAGCGCGACGGCGA.

EMSA. Nuclear extracts from chicken embryonic transfected withcorresponding expression plasmids(Sox9-Ets1-, cMyb- or control-pCIH2BRFP) were obtained using standard hypotonic buffer (10 mM HEPESpH7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 1X Complete EDTA-FreeProtease Inhibitors, 0.2 mM PMSF) to isolate the nuclei and extractionbuffer (20 mM HEPES pH7.9, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25%(v/v) glycerol, 1 mM DTT, 1X Complete EDTA-Free Protease Inhibitors) toobtain nuclear extracts. Binding reactions and gel shift detection wasperformed using LightShift Chemiluminescent EMSA Kit (Thermo FisherScientific, Rockford, Ill.) following manufacturer's instructions.

Pull-Down Assays. Pulldown binding assays were performed usingstreptavidin Dynal beads (Invitrogen) and the same biotin-labeledSox10E2 subfragments as in EMSA, carrying a single biotin tag. Dynalstreptavidin beads (Invitrogen) were coated according to manufacturer'sinstructions, using equimolar quantities of the fragment labeled oneither 5′ or 3′ end. 400-500 μg of embryonic nuclear proteins (extractedfrom cephalic regions of chicken embryos at stage HH9-HH2) in finalvolume of ˜800 μl of binding buffer (12% glycerol, 12 mM HEPES pH 7.9, 4mM Tris pH 7.9, 150 mM KCl, 1 mM EDTA, 1 mM DTE, and 0.1 μg/μlpoly(dI-dC). These were pre-exhausted using ˜1.5 mg of streptavidinDynal beads and distributed among 4 Dynal bead preps: 2 coated withspecific DNA fragment, one with scrambled control and one without DNA.Binding reactions were allowed to proceed for 30′ at RT, and weresubsequently washed 4 times with the binding buffer (only 1st washcontained poly(dI-dC)). After the 4^(th) wash, the beads weretransferred to a new tube, bound proteins eluted in 30 μl of 50 mM TrispH 7.5, 100 mM NaCl, 5 mM EDTA, 1× Protease Inhibitors, 0.1% SDS andanalyzed by Western blot using Sox9, Ets1 and cMyb antibodies.

Ets1, cMyb and Sox9 antibodies recognize cross-linked endogenouscorresponding proteins. (FIG. 15A, 15B) Western blots, using Sox9 andcMyb antibodies on cross-linked or cross-linked/sonicated materialperformed under moderate denaturing conditions. Specific bands werenoted at ˜120 kDa for Sox9 and at ˜80 kDa and 120 kDa for cMyb. SimilarSox9 and cMyb protein bands were detected after pull-down assays. 120kDa cMyb protein was also bound to cMyb scrambled control (Myb CO) with70% homology threshold (FIG. 15D). Partial denaturating conditions showthat other less prominent bands likely represent complex associationsrather than Sox9 or cMyb bands, as they are less prominent in thesonicated condition. (FIG. 15C) ChIP using Ets1 antibody, followed bywestern blot using the same target antibody. Detection was performedusing IP-Western kit (GenScript, Piscataway, N.J.) and shows severalbands in cross-linked and sonicated input, under moderate denaturatingconditions, with a single Ets1—precipitated band (˜arrow) detected inthe ChIP and input lanes, but not in samples precipitated with IgGantibody. IP—Immunoprecipitated fraction, SN—1^(st) supernatant, IgGIP—Immunoprecipitation with normal rabbit IgGs (negative control),M2—subfragment of Sox10E2 enhancer containing N-terminal Myb site,M11/12—subfragment of Sox10E2 enhancer containing C-terminal Myb site,Myb CO—scrambled control fragment containing no Myb sites with >70%homology, -DNA—control pulldown using streptavidin magnetic beads only,without DNA fragment.

Chromatin immunoprecipitation with Sox9, Ets1 and cMyb antibodies. ForChIP, chromatin was prepared from cranial regions of 8-12 somite chickenembryos and immuno-precipitated using Ets1(sc-350; Santa Cruz), Sox9(ab71762, Abcam and rabbit polyclonal, Dr. M. Wegner) and cMybantibodies (Karafiat et al. (2005) Cell Mol Life Sci 62(21):2516-2525)with normal rabbit IgGs (sc-2027, Santa Cruz; ab27478, Abcam) asnegative controls. The protocol was adapted from (Buchholz et al (2006)Methods in Signal Transduction, eds Whitman M & sater Ak (CRC Press),257-271; Dahl and Collas (2008) Nat Protoc 3(6): 1032-1045).

For each preparation of nuclei, cranial regions from 20 stage 8-12somite embryos were dissected in Ringer's solution and transferred to 1ml isotonic buffer (0.5% Triton X-100, 10 mM Tris-HCl, pH 7.5, 3 mMCaCl2, 0.25 M sucrose, protease inhibitor tablet (Complete ProteaseInhibitor EDTA-free, Roche), 1 mM DTT, and 0.2 mM PMSF) on ice (adaptedfrom Buchholz et al. 2006). Tissue was homogenized using Douncehomogenizer and cells cross-linked by adding formaldehyde to a finalconcentration of 1% and nutated for 10′ at room temperature. Glycine(final concentration of 125 mM) was added to stop the cross-linkingreaction and solution was incubated by nutation for 5′ at RT. Thecross-linked cells were washed 3 times and cell pellets were either snapfrozen in liquid nitrogen and kept at −80° C. until the ChIP procedure.Preparations were kept up to a month without altering the quality ofresults. The pellets were re-suspended in isotonic buffer and nucleiisolated using Dounce homogenizer washed and lysed in SDS lysis buffer(1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) for 10′-1 h. The lysate wasthen diluted three-fold with ChIP dilution buffer (0.01% SDS, 1.2 mMEDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1 mM DTT, 0.4 mM PMSF, andprotease inhibitors) and ½ of chromatin (420 ul) was sonicated usingMisonix 4000 sonicator at following settings: Amp 3, 10 consecutivecycles of 30″ sonication each with 1′ pause in between. Triton X wasadded to the sonicated material to a final concentration of ˜1%,chromatin was cleared by centrifugation, diluted 3-4 times with ChIPDilution Buffer w/1.1% Triton X-100 and was distributed between 2-3antibody/bead complexes (400 ul each) and incubated ON at 4° C. 50 ul ofthe chromatin preparation was conserved at −80° C. as the inputfraction. Antibody/magnetic bead were prepared as per Young protocol.Post-immunoprecipitation washes were performed using RIPA wash buffer(50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7%Na-Deoxycholate). The complexes were then washed with Tris-EDTA/NaCl (10mM Tris-HCl, 1 mM EDTA pH 8.0, 50 mM NaCl) for 5′ and transferred to anew chilled tube, prior to last separation (Dahl and Collas, 2008). Thechromatin was eluted in elution buffer (1% SDS, 10 mM EDTA, 50 mMTris-HCl, pH 8.0) and cross-link reversed overnight by incubation at 65°C. The samples were consecutively treated with RNase A (0.2 mg/ml) andthen Proteinase K (0.2 mg/ml), extracted with Phenol/Chloroform/IsoamylAlcohol, precipitated and resuspended in 50 ul of 10 mM Tris pH 8.0.Real-time PCR reactions were performed in a 96-well plate ABI7000 QPCRmachine. Reactions were set up using SybrGreen (Biorad), 450 μM of each(forward and reverse primer) and 1 ul of each ChIP reaction or 1:100-200dilution of Input fraction. The ΔΔC_(t) method was used forquantification and calculations performed according to ChIP-qPCR DataAnalysis instructions (SupperArray, Bioscience Corporation). In order toselect suitable negative control primers, large regions (potentiallycorresponding to genomic deserts) of chromosome 1 were surveyed. BecauseEts1 binding sites are present in large proportion of the chickengenome. non-specific binding was a concern; therefore, 8-10 differentsets of primers were tested.

The primers presented in FIG. 13M corresponding to Sox10E2 fragmentwere:

Sox10E2_1 (SEQ ID NO: 152) 5′-TGCTCCTGCTGCTTATCA-3′; Sox10E2_1 rev(SEQ ID NO: 153) 5′-ATCAGCTCCACTGCACAT-3′; Sox10E2_2 (SEQ ID NO: 154)5′-TGATAAGCAGCAGGAGCA-3′; Sox10E2_2 rev (SEQ ID NO: 155)5′-TGAGCAGGT-TGCTGTGGAAA-3′.

Control primer sets that amplify negative control region situated on thesame chromosome as Sox10 locus were as follows:

negcont_1 (SEQ ID NO: 156) 5′-TCGGATTTTAATGGGCTCAG-3; negcont_1 rev(SEQ ID NO: 157) 5′-CCGCAGATAGTTCTGCATCA-3′; negcont_2 (SEQ ID NO: 158)5′-GGTTGGATTTCCAGTCTCCA-3′; negcont_2 rev (SEQ ID NO: 159)5′-TGTTTTGCTG-GACAATCTGC-3′.

Ets1, cMyb and Sox9 bind directly to Sox10E2 enhancer element in vivo.Direct binding of Ets1, cMyb and Sox9 transcription factors to theSox10E2 regulatory element driving the onset of Sox10 expression in thedelaminating neural crest was assessed using chromatinimmunoprecipitation (qChIP). (FIG. 16A) shows qChIP positive controlusing antibody against H3K36me3, chromatin mark generally associatedwith active gene transcription. Chromatin immunoprecipitation (ChIP)shows high occupancy of H3K36me3 mark in proximity of thetranscriptional start site (TSS) of Sox10 and control active β-actinlocus. Relative enrichment over input using specific and negativecontrol antibodies (normal rabbit IgGs) are presented. FIG. 16 b showsqChIP results obtained using specific target antibodies (Sox9, Ets1 andcMyb) to precipitate chromatin regions specifically bound by thosefactors. The results presented as relative enrichment over input usingspecific or negative control antibody (normal IgGs) were obtained byquantitative PCR where the same amount of DNA from each pulldown wasused in a separate reaction and two primer sets specific with Sox10E2region (red bars) and two primer sets within negative non-bound region(grey bars). FIG. 16 c shows enrichment is expressed relative to inputDNA using the same amount of DNA in the QPCR reaction for each ChromatinIP. Enrichment of the specific factors (Ets1, cMyb or Sox9) at Sox10E2enhancer region is presented as red bars, enrichment of specific factorat negative control regions is presented in blue. Background levels(mean enrichment from control antibodies) at enhancer and negativecontrol regions are shown as pale red or pale blue bars, respectively.FIG. 16 d shows means and standard deviations of relative enrichment(fold over negative control antibody) are presented from 3-4 independentexperiments for each specific antibody used. Green bars representenrichment at Sox10E2, grey bars at negative control regions.

Over Expression Constructs

Open reading frames of chick Sox9, cMyb and Ets1 genes were amplifiedfrom a full length cDNA clones or chicken cDNA (Sox9 cDNA clone was agift from Yi-Chuan Cheng, Ets1 clone was isolated from the stage 8-12somites chicken macroarrayed cDNA library constructed by Laura Gammill(Kelsh R N (2006) Bioessays 28(8):788-798) and cloned into XhoI/EcoRV orXhoI/ClaI digested pCI H2B-RFP expression vector. The Sox9 and Ets1rescue constructs were generated from expression constructs by PCR,using primers carrying mutations within the morpholino target sites thatdo not alter the amino acid sequence of the recombinant proteins. Thefollowing primers were used:

Sox9_5′ (SEQ ID NO: 160) 5′-AATTCTCGAGGCCACCTGCTCAAGGGCTACGACTGG-3′ andSox9_3′ (SEQ ID NO: 161) 5′-ATTAGATATCTTTAAGGCCGGGTGAGCTGC-3′; Ets1_5′(SEQ ID NO: 162) 5′-AATACTCGAGGGCCTCAACCATGAAGGCGGCGGTGGA-3′ and Ets1_3′(SEQ ID NO: 163) 5′-ATTAGATATCTCACTCATCAGCATCTGGCTTG-3′; cMyb_5′(SEQ ID NO: 164) 5′-ATTACTCGAGgccaccATGGCCCGGAGAC; cMyb_3′,(SEQ ID NO: 165) 5′-ATTAATCGATTCACATCACCAGAGTCC; Sox9mut_5′(SEQ ID NO: 166) 5′-ATTACTCGAGgccaccATGAActTgtTgGAtCCCTTCATGAAAATGAC;Ets1mut_5′, (SEQ ID NO: 167)5′-ATTACTCGAGTCAACCATGAAaGCtGCcGTcGAttTaAAaCCCACCCTGACCATCA.

Transcriptional inputs into the Sox10E2 regulatory region. FIG. 8 ashows EGFP pattern of expression in neural crest cells driven by theintact Sox10E2 regulatory region. FIGS. 8B, 8C show EGFP expression isabolished in cranial neural crest when binding motifs for putativeupstream regulators, SoxE and a pair of Myb are mutated (SoxE, M8 inFIG. 8B and Myb, M2/M12 in FIG. 8C). FIG. 8D shows a mutation of asingle Myb binding site mildly decreases EGFP reporter expression whileFIG. 8 e shows that a NFkB1 mutation does not affect reporter signal.

Comparative Genomic Analyses

To identify highly conserved genomic regions, the ECR Browser softwarewas employed. Chicken, zebrafish, Xenopus, opossum, mouse, chimpanzee,dog, rat and human genomic sequences were downloaded using UCSC genomebrowser. Following instructions available on the ECR website, thesesequences were computationally compared from between all the species,with conservation parameters set to 70-80%. The “zoom in” feature, builtin the program, was used to closely analyze the sequence conservation byincreasing the threshold up to 90% and using different window sizesranging from 20 bp to 50 bp. To search for putative binding motifs, weused the jaspar_core database from Jaspar and the P-Match programavailable through Transfac database. Briefly, the 264 bp-long sequenceof Sox10E2 genomic fragment was uploaded into these programs usingdesired parameters and the programs returned potential binding motifsbased on the position weight matrix (PWM) score. Simultaneously, usingrVista 2.0, Sox10E2 sequence was aligned and compared to thecorresponding mouse sequence to screen the latter sequence for conservedputative binding motifs identified in chicken by either Jaspar, Transfacor both search engines. We used the position weight matrix (PWM) score,a value given to a site based on the distribution frequency of each baseat each position (Sauka-Spengler T & Bronner-Fraser M (2008) Nat Rev MolCell Biol 9(7): 557-568) to determine the probability of binding score.This was then used to guide mutational analysis, since, as with anyinformatics approach, there are several caveats: 1) this only predictsmotifs for factors with known consensus sites; 2) not all functionalsites have high PWM scores since they can differ greatly from consensus(Antonellis et al. (2006) Hum Mol Genet 15(2): 259-271) and 3) not allsites with high PWM are functional.

Cell Death and Proliferation Assays

Histology. Embryos electroporated unilaterally with single or triplemorpholino (@3 mM concentration) were fixed in 4% paraformaldehyde anddehydrated to 100% methanol. After re-hydration, embryos werecryoprotected in 15% sucrose, equilibrated in 15% sucrose/7.5% gelatin,embedded in 20% gelatin and sectioned at 10 um using Micron cryostat.

TUNEL reaction. The slides were first washed twice in PBS at 42° C. for10′ to remove gelatin, followed by 3-4 10′ washes in PBST (PBS+0.5%Triton X-100) at RT. After 10′ incubation in Permeabilization solution(0.1% sodium Citrate, 0.5% Triton in PBS) and two PBST rinses, theslides were incubated in Tunel reaction mix (In Situ Cell DeathDetection Kit, TMR red, Roche). Labeling solution was first diluted 10times with Tunel buffer (30 mM Tris pH 7.2, 140 mM Sodium cacodylate, 1mM CoCl₂) and then combined with enzyme mix as per manufacturer'sinstructions (1 part enzyme+9 parts label). A positive control slide,pre-treated with DNAse I (2 ul of 10 U/ul stock in 100 ul of DNasebuffer: 10 mM CaCl₂, 40 mM Tris Cl pH7.4, 10 mM MgCl₂, mM NaCl, for 1 hat RT) was prepared in advance, rinsed with 2 mM EDTA in PBST to quenchDNase activity, washed twice in PBST and stained with Tunel reaction mixas well. Negative control slide was incubated in Tunel labeling mix,without TdT enzyme. The labeling was performed in the dark, for 4 h in ahumidified chamber at 37° C. The slides were then washed 3×PBST for atleast 15 min each time, the positive control slide was rinsed in aseparate container.

Immunostaining. The slides were incubated in blocking buffer (10% donkeyserum in PBST) for 1 hour followed by primary antibodies diluted inblocking buffer overnight at 4° C., (1:1000 rabbit antiPh3(Phospho-Histone 3) and 1:800 Alexa 488 goat anti FITC). Slides werethen rinsed 3-4 times in PBST, 30 min each wash. Secondary antibody wasdiluted in PBST or blocking buffer and applied for 1 hour at RT. As theTUNEL staining is red (TMR red), we used 1:1000 Alexa 350 goatanti-Rabbit (blue) to detect antiPh3 and FITC (morpholino) was labeledin green. All consecutive sections from the cranial region were countedand number of Ph3-(and Tunel-) positive cells within the neural fold wascompared between morpholino-ed and control sides for individual, tripleand control morpholinos. We present the mean value ofelectroporated/control side ratio for triple and control morpholinos.The statistical values were calculated using unpaired student t-test.

Ets1 and cMyb transcription factors are necessary for activation ofSox10E2 regulatory element. FIG. 9A shows control morpholino (MO)(right; red) has no effect on Sox10E2-driven Cherry (FIG. 9B; green)compared to non-electroporated (left) side. FIG. 9D shows cMyb MOsignificantly reduces, whereas Ets1 MO (FIG. 9G) abolishesSox10E2-driven Cherry expression, (FIGS. 9E, 9H, respectively. FIGS. 9C,9F, 9I are merged images of FIGS. 9A/B, 9D/E and 9G/H, respectively).White dotted lines=midline. Green/red channels inverted for consistency.

In situ hybridization is shown in FIGS. 9J-9N. FIGS. 9J-9L show thatendogenous cMyb, Sox9 and Ets1 expression precedes that of Sox10,consistent with being upstream regulators. At HH6, cMyb is expressedwithin the neural plate border (FIG. 9J) and confined to dorsal neuralfolds containing CNC by HH8 (FIGS. 9K; 9K′; arrowheads). At HH10, cMybis observed in migrating neural crest (FIG. 9L and section at dottedline, FIG. 9L′ arrows). At HH8, prior to Sox10 onset, Sox9 (FIG. 9M) andEts1 (FIG. 9N) are expressed by presumptive cranial neural crest in thedorsal neural tube.

Morpholino-mediated Knock-down Experiments

Morpholino-mediated knock-down experiments were performed by injectingthe translation-blocking, FITC-labelled morpholino antisenseoligonucleotides in one half of the epiblast (right to the primitivestreak) or, in some cases, by double electroporations to differentiallytransfect each half of the embryo ex-ovo. For Sox10E2, doubleelectroporations were performed by introducing each morpholino combinedwith the Sox10E2-Cherry plasmid on the right side and the Sox10E2-Cherryreporter only on the left side of the embryo. Morpholinos for knockdownsin conjunction with SOX10E2-EGFP were obtained from Gene Tools(Philomath, Oreg.) and their sequences are as follows:

Ets1 (SEQ ID NO: 168) 5′-GCTTCAGGTCCACCGCCGCCTTCAT-3; cMyb(SEQ ID NO: 169) 5′-ATGGCCGCGAGCTCCGCGTGCAGAT-3′; Sox9 (SEQ ID NO: 170)5′-GGGTCTAGGAGATTCATGCGAGAAA-3′; Control (SEQ ID NO: 171)5′-ATGGCCTCGGAGCTGGAGAGCCTCA-3′.The final molar concentration of each morpholino oligonucleotide usedwas 725 nM.

Morpholino-mediated Sox9 knock-down significantly reduces Sox10E2regulatory activity. FIG. 10A shows FITC-labeled control morpholino (inred) does not affect Sox10E2 driven Cherry expression in FIG. 10B(green). FIG. 10C shows a merged image of FIGS. 10A and 10B, revealingwith control morpholino (red) with Sox10E2 driving expression of Cherry(green). FIG. 10D shows Sox9 FITC-labeled morpholino (red) stronglyreduces Sox10E2 driven Cherry expression as shown in FIG. 10E (green,white arrow). FIG. 10F shows a merged image of 10D and 10E. Embryos wereelectroporated on the right side only. The images were pseudo-coloredusing Photoshop, with green and red channels inverted for consistency,indicative of reporter expression.

Sox9, cMyb and Ets1 are required for endogenous Sox10 expression indelaminating neural crest cells. FIGS. 11A-1M show HH8+ embryos withunilateral electroporation of Sox9 (FIGS. 11A, 11E), cMyb (FIGS. 11B,11F) and Ets1 (FIGS. 11C, 11G) morpholinos (MO) show significantdecrease in endogenous Sox10 expression in delaminating CNC compared tonon-electroporated side, whereas control MO(FIGS. 11L, 11M) has noeffect. Co-electroporation of Sox9, cMyb, Ets1 MOs completely abolishesendogenous Sox10 expression (10D,10H). Showing specificity, the effectis rescued by co-electroporation with corresponding expressionconstruct: Sox9 MO+Sox9 DNA(10I), cMyb MO+cMyb DNA(10J) or Ets1 MO+Ets1DNA(10K). Statistical relevance by chi-squared test of MOs on Sox10expression was p<0.02; of rescues was p<0.03(Sox9; Ets1) andp≦0.04(cMyb).

Electroporation of Ets1, cMyb, Sox9 and control morpholinos does notaffect cell proliferation and does not induce apoptotic cell death.FIGS. 12A, 12 b 12B show embryos electroporated with FITC-labeled triple(Ets1, cMyb and Sox9) or control morpholinos. FIGS. 12C, 12D showembryos sectioned, followed by TUNEL staining. FIGS. 12E, 12F showanti-Phospho-Histone H3 (Ph3) antibody staining of sections. FIGS. 12G,12H show overlays of FITC, TUNEL and Ph3 staining presented in (FIGS.12A, 12C. 12E) and (FIGS. 12B, 12D, 12F), respectively. All consecutivesections from the cranial region were counted and the number of Ph3-(and Tunel-) positive cells within the neural fold was compared betweenmorpholino-ed and control sides. Mean value and standard deviation ofelectroporated/control side ratio from four independent embryos arepresented (FIG. 12I shows TUNEL, red bars; FIG. 12J shows Ph3, bluebars). The statistical calculations performed using unpaired studentt-test show no statistically significant differences in cell death orproliferation counts between electroporated and control sides of embryosreceiving either three specific morpholinos or control (at 3 mM).

Binding motifs for SoxE, Ets and Myb within Sox10E2 enhancer need to befunctional in order for ectopic reporter expression to occur whenmisexpressing Sox9, cMyb and/or Ets1, individually or in combination.FIG. 14D shows ectopic SOX10E2-activated EGFP expression in theextraembryonic region, ectoderm cells (arrowheads) and along the neuraltube when Sox9-pCI H2B-RFP, Ets1-pCI H2B-RFP and cMyb-pCI H2B-RFP (red)are simultaneously over-expressed as shown in FIG. 14A. FIG. 14B showsthat combined Sox9 and cMyb misexpression fails to activate ectopic EGFPexpression through a mutated Sox10E2 regulatory region (M9) that lacksan Ets binding motif (FIG. 14E). Whereas, overexpressing Sox9 and Ets(FIG. 14C) simultaneously can activate weak reporter expression (arrows)through a mutated Sox100E2 lacking one Myb (M12) binding motif (FIG.14F).

Sox9, cMyb and Ets1 overexpression ectopically induces Sox10E2regulatory activity. EGFP is observed in migrating crest and oticvesicle (OV) when Sox10E2-EGFP is co-electroporated with controlplasmid, pCI H2B-RFP (FIGS. 13A, 13F). Overexpression of either Sox9(FIGS. 13B, 13G), Ets1(FIGS. 13C, 13H) or cMyb (FIGS. 13D, 13I)ectopically activates Sox10E2-driven EGFP expression in extraembryonicectoderm (white arrows). In FIG. 13H, arrowheads show EGFP expression inposterior neural tube. Misexpression of Ets1(FIG. 10E) fails to activateectopic EGFP expression (FIG. 13J, arrows) in a mutated Sox10E2construct lacking an Ets binding motif(M9).

EMSA shows a clear band shift (FIG. 13K, white arrowhead) when nuclearextracts containing overexpressed Sox9, Ets1 or cMyb proteins arecombined with Sox10E2 subfragments, M11, M9 and M2, respectively (1^(st)lane). This binding is outcompeted when excess non-labeled probe isadded (2^(nd) lane) and absent from nuclear extracts from controlplasmid-transfected cells (3^(rd) lane). Biotinylated Sox10E2subfragments (M8,M11-Sox9, M4,M9-Ets1 and M2,M12-cMyb), as well asscrambled control fragments and non-coated Dynal streptavidin beads,used as bait in a DNA pulldown assay show specific transcription factorbinding as analyzed on a Western blot in FIG. 13L. FIG. 13M shows directbinding of Ets1, cMyb and Sox9 to the Sox10E2 enhancer element in vivoas assessed by qChIP. Binding to Sox10E2 (red bars) or control region(grey bars) was assessed with two primer sets for each region andexpressed as relative enrichment of target over control antibody; graphreflects mean±SD from a representative experiment. qChIP was performed3-4 times for each factor. Enrichment relative to input DNA from allindependent experiments is shown in FIGS. 16A-D.

Short-hairpin RNA Vectors

Vector design and modification. The vectors in FIGS. 24A, 24B weregenerated through sequential generations of adaptation and modification.Enhancers are cloned into a KpnI/BglII multiple cloning site and shorthairpins cloned into a unique EcoR1/XhoI sites. In FIG. 24A, the NCenhancer sequence (SEQ ID NO: 4) is shown. The full sequence schematicfor the vector in FIG. 24B is shown in Appendix (SEQ ID NO: 172). Thegeneral vector designs are shown in FIG. 25 wherein Vector 1 concept isthe vector of FIG. 24A and Vector 2 concept is the vector of FIG. 24B.Integration sites for genomic insertion can be provided in either vectorproviding the same knockdown results.

Short hairpin design and amplification. Oligonucleotides (oligos) wereselected using software designed by the Hannon laboratory at Cold SpringHarbor Laboratory, and the 90-mer ordered from IDT (Integrated DNATechnologies Inc., San Diego 92121, USA). These were amplified andEcoR1/XhoI sites added using the primers: SH2fwd (SEQ ID NO: 173)(5′-GATGGCTG-CTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′) and SH2reverse (SEQID NO: 174) (5′-GTCTAGAGGAATTCCGAGGCAGTAGGCA-3′). This was performedusing the following PCR reaction: Thermopol Buffer (1× final cone.)Mg2SO4 2 mM. DMSO 5%, dNTPs 200 uM, Template oligo 100 ng, ForwardPrimer 0.5 uM, Reverse Primer 0.5 uM, VENT Polymerase 1 U/reaction. Theconditions used are: 1) 94° C. 5 mins, 2) 94° C. 30 seconds, 3) 54° C.30 seconds, 4) 75° C. 30 seconds, cycles 2-4 repeated 12 cycles; 5) 75°C. 2 mins.

Electroporation and analysis for short-hairpin miRNA vectors. Unlessotherwise stated, embryos were electroporated at HH3+−HH4 (Hamburger &Hamilton 1951) and placed in EC culture (Chapman et al. 2001, Dev. Dyn.220(3): 284-289). The left side only of the neural plate waselectroporated in all cases (unilateral electroporation). Embryos wereelectroporated with 5×7V of a pulsed square current (50 ms ON 100 msOFF) and incubated at 37 degC, 5% CO2 until they reached the requiredsomite number. For QPCR, embryos were bisected down the midline intoelectroporated and non-electroporated sides and cDNA synthesis/QPCR wasperformed separately on each half. Data fromelectroporated/non-electroporated for targeted and control (RFP) embryoswere compared (FIG. 26). Electroporation at 4 mg/ml (FIG. 26) givesefficient knockdown of target gene during the period the enhancer isactive. When unilateral electroporatoration was unfeasible, embryos werebilaterally electroporated whole embryos used for QPCR with targetedversus RFP results presented. For experiments using the MSX-shFoxD3 andSpalt-4-shPax3 vectors, embryos were electroporated at HH3 at a pointprior to ingression of the mesoderm. This allows sufficient time fortranscription of short hairpins to begin prior to expression of thetargeted gene. In situ hybridization as shown in FIGS. 26A, 26B wereperformed as described herein.

Neural Crest Cell Isolation/Dissociation

Electroporate the reporter construct of interest (enhancer drivingfluorescent protein) or use the transgenic mouse expressing thefluorescent reporter under the control of neural crest enhancer. Dissectthe region of interest. Collect tissue in Ringer's and keep on ice. Thawa vial of each: dispase (prepared in DMEM+Hepes at a cc of 1.5 mg/ml)and trypsin (Trypsin-EDTA: 1×0.05% Trypsin, 0.53 mM EDTA in HBSS). Usethe 37° Celsius (C) water bath for 5 min. Treat the dissected tissuewith 2 ml of dispase for 10 min at 37° C. Use the 15 ml falcon tube, andtriturate (using 2 ml plastic sterile pipets) and incubate foradditional 5 min at 37° C. Before trituration, pipet up and down media+10% FBS to coat the pipet and prevent cells from sticking to it.Triturate, add 40 ul of Trypsin and incubate for 1 min. Stop thereaction by adding 10 mls DMEM, 10% FBS. Centrifuge at 1000 ref for 10min. Remove the supernatant, re-suspend the cells in 1 ml of Hankssolution (w/o phenol red+2.5 mg/ml BSA fraction V). Place the 40micrometer filter on a 50 ml falcon tube. Pass the 1 ml+ cells throughthe filter. Take out 10 ul of Hanks+ cells and add 10 ul of trypan blue.Count the cells using hemocytometer: (# of cells×dilutionfactor)×2×10^4=# of cells/ml. Take a small sample and look underfluorescence microscope. Centrifuge at 1000 ref for 10 min. Take out˜600 ul of Hanks solution and leave cells re-suspended in the remaining300-350 ul of hanks solution. Transfer the cells in Hanks solution to a5 mls falcon tube. Prepare a few 1.5 eppendorf tubes (4 per sample to besorted) with approximately 1 ml of media (DMEM+10% FBS) to collectsorted cells. Run the cell suspension through the FACS(Fluorescence-Activated Cell Sorting) machine and collect isolatedneural crest cells that express fluorescent marker under the control ofneural crest enhancers. For incubations at 37° C. use a water bath. Toreduce the stickiness of the cells, due to accidental nuclear lysis, theprep can be treated with DNAse for a short time. This treatment shouldbe avoided if whole cell extracts are to be used in DNA sensitiveapplications.

In summary, DNA enhancer sequences are provided for use in constructs toidentify early stage embryonic cells. The enhancer sequences can be usedin parallel with short-hairpin RNA in a vector construct forendogenously regulated gene knockdowns. The disclosed enhancer sequencescan be used to isolate a selected population of early stage embryoniccells.

All references cited in the application are incorporated in theirentirety as if explicitly recited herein, particularly all referencesdirected to methodologies of synthesis.

What is claimed is:
 1. An isolated nucleic acid sequence consisting ofSEQ ID NO:
 4. 2. A nucleic acid vector for down-regulating geneexpression comprising: a short-hairpin RNA sequence undertranscriptional control of at least one enhancer sequence having thesequence as set forth in SEQ ID NO:
 4. 3. An expression vectorcomprising the sequence of SEQ ID NO: 4 and a nucleic acid sequenceencoding a reporter protein.
 4. The expression vector of claim 3,wherein the reporter protein is a fluorescent protein.
 5. The expressionvector of claim 3, wherein the expression vector is a viral vector.
 6. Amethod of isolating mammalian cells expressing FoxD3, the methodcomprising: introducing to a population of isolated mammalian cells anexpression vector comprising the sequence of SEQ ID NO: 4 and a nucleicacid sequence encoding a reporter protein; and identifying introducedcells that produce the reporter protein.
 7. The method of claim 6,wherein the reporter protein is a fluorescent protein.
 8. The method ofclaim 6, further comprising isolating the cells expressing the reporterprotein.
 9. The method of claim 6, wherein the isolating is performed byflow cytometry.